Estimate of the Frequency of Human Immunodeficiency Virus Type 1 Protease Inhibitor Resistance within Unselected Virus Populations In Vitro

The frequency of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants in virus populations not previously exposed to drug was determined in vitro by using HIV-1_RF and the protease inhibitor SC-55389A. Two variants with single mutations responsible for drug resistance (V82A and N88S) were quantifiably isolated after only one round of replication, yielding a crude frequency estimate of at least 1 SC-55389A-resistant variant per 3.5 × 10⁵ wild-type infectious units.     

Characterization of a novel human immunodeficiency virus type 1 protease inhibitor, A-790742

A-790742 is a potent human immunodeficiency virus type 1 (HIV-1) protease inhibitor, with 50% effective concentrations ranging from 2 to 7 nM against wild-type HIV-1. The activity of this compound is lowered by approximately sevenfold in the presence of 50% human serum. A-790742 maintained potent antiviral activity against lopinavir-resistant variants generated in vitro as well as against a panel of molecular clones containing proteases derived from HIV-1 patient isolates with multiple protease mutations. During in vitro selection, A-790742 selected two primary mutations (V82L and I84V) along with L23I, L33F, K45I, A71V/A, and V77I in the pNL4-3 background and two other mutations (A71V and V82G) accompanied by M46I and L63P in the HIV-1 RF background. HIV-1 pNL4-3 clones with a single V82L or I84V mutation were phenotypically resistant to A-790742 and ritonavir. Taking these results together, A-790742 displays a favorable anti-HIV-1 profile against both the wild type and a large number of mutants resistant to other protease inhibitors. The selection of the uncommon V82L and V82G mutations in protease by A-790742 suggests the potential for an advantageous resistance profile with this protease inhibitor.     

Evolution of primary protease inhibitor resistance mutations during protease inhibitor salvage therapy

In order to track the evolution of primary protease inhibitor (PI) resistance mutations in human immunodeficiency virus type 1 (HIV-1) isolates, baseline and follow-up protease sequences were obtained from patients undergoing salvage PI therapy who presented initially with isolates containing a single primary PI resistance mutation. Among 78 patients meeting study selection criteria, baseline primary PI resistance mutations included L90M (42% of patients), V82A/F/T (27%), D30N (21%), G48V (6%), and I84V (4%). Despite the switching of treatment to a new PI, primary PI resistance mutations present at the baseline persisted in 66 of 78 (85%) patients. D30N persisted less frequently than L90M (50% versus 100%, respectively; P < 0.001) and V82A/F/T (50% versus 81%, respectively; P = 0.05). HIV-1 isolates from 38 (49%) patients failing PI salvage therapy developed new primary PI resistance mutations including L90M, I84V, V82A, and G48V. Common combinations of primary and secondary PI resistance mutations after salvage therapy included mutations at amino acid positions 10, 82, and 46 and/or 54 in 16 patients; 10, 90, and 71 and/or 73 in 14 patients; 10, 73, 84, 90, and 46 and/or 54 in 5 patients; 10, 48, and 82 in 5 patients; and 30, 88 and 90 in 5 patients. In summary, during salvage PI therapy, most HIV-1 isolates with a single primary PI resistance mutation maintained their original mutations, and 49% developed additional primary PI resistance mutations. The persistence of L90M, V82A/F/T, G48V, and I84V during salvage therapy suggests that these mutations play a role in clinical resistance to multiple PIs.     

Mutation D30N is not preferentially selected by human immunodeficiency virus type 1 subtype C in the development of resistance to nelfinavir

Differences in baseline polymorphisms between subtypes may result in development of diverse mutational pathways during antiretroviral treatment. We compared drug resistance in patients with human immunodeficiency virus subtype C (referred to herein as "subtype-C-infected patients") versus subtype-B-infected patients following protease inhibitor (PI) therapy. Genotype, phenotype, and replication capacity (Phenosense; Virologic) were determined. We evaluated 159 subtype-C- and 65 subtype-B-infected patients failing first PI treatment. Following nelfinavir treatment, the unique nelfinavir mutation D30N was substantially less frequent in C (7%) than in B (23%; P = 0.03) while L90M was similar (P < 0.5). Significant differences were found in the rates of M36I (98 and 36%), L63P (35 and 59%), A71V (3 and 32%), V77I (0 and 36%), and I93L (91 and 32%) (0.0001 < P < 0.05) in C and B, respectively. Other mutations were L10I/V, K20R, M46I, V82A/I, I84V, N88D, and N88S. Subtype C samples with mutation D30N showed a 50% inhibitory concentration (IC(50)) change in susceptibility to nelfinavir only. Other mutations increased IC(50) correlates to all PIs. Following accumulation of mutations, replication capacity of the C virus was reduced from 43% +/- 22% to 22% +/- 15% (P = 0.04). We confirmed the selective nature of the D30N mutation in C, and the broader cross-resistance of other common protease inhibitor mutations. The rates at which these mutational pathways develop differ in C and subtype-B-infected patients failing therapy, possibly due to the differential impact of baseline polymorphisms. Because mutation D30N is not preferentially selected in nelfinavir-treated subtype-C-infected patients, as it is in those infected with subtype B, the consideration of using this drug initially to preserve future protease inhibitor options is less relevant for subtype-C-infected patients.     

Sensitivity of the ViroSeq HIV-1 genotyping system for detection of the K103N resistance mutation in HIV-1 subtypes A, C, and D

The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping methods detect antiretroviral drug resistance mutations present in the major viral population of a test sample. These assays also detect some mutations in viral variants that are present as mixtures. We compared detection of the K103N nevirapine resistance mutation using ViroSeq and a sensitive, quantitative point mutation assay, LigAmp. The LigAmp assay measured the percentage of K103N-containing variants in the viral population (percentage of K103N). We analyzed 305 samples with HIV-1 subtypes A, C, and D collected from African women after nevirapine administration. ViroSeq detected K103N in 100% of samples with >20% K103N, 77.8% of samples with 10 to 20% K103N, 71.4% of samples with 5 to 10% K103N, and 16.9% of samples with 1 to 5% K103N. The sensitivity of ViroSeq for detection of K103N was similar for subtypes A, C, and D. These data indicate that the ViroSeq system reliably detects the K103N mutation at levels above 20% and frequently detects the mutation at lower levels. Further studies are needed to compare the sensitivity of different assays for detection of HIV-1 drug resistance mutations and to determine the clinical relevance of HIV-1 minority variants.     

New reporter cell line to evaluate the sequential emergence of multiple human cytomegalovirus mutations during in vitro drug exposure

We developed a new reporter cell line for human cytomegalovirus (HCMV) drug susceptibility testing. This cell line was obtained by incorporating the luciferase reporter gene under the control of an HCMV-specific promoter into the genome of astrocytoma cells (U373MG). We then used our reporter cell line to evaluate phenotypic changes conferred by the sequential emergence of HCMV UL54 and UL97 mutations following long-term drug exposure. The laboratory strain AD169 was passaged in the presence of increasing concentrations of ganciclovir (one viral line) or foscarnet (two viral lines). Resistant viruses were plaque purified at five different concentrations of ganciclovir and at three different concentrations of foscarnet. In addition to the previously described M460I and L595S UL97 mutations and the L545S and V812L UL54 mutations, exposition to ganciclovir (up to 3,000 microM) resulted in the selection of two unreported UL54 mutations (P829S and D879G). Passages in the presence of foscarnet (up to 3,000 microM) resulted in the selection of seven not previously described UL54 mutations (K500N, T552N, S585A, N757K, L802V, L926V, and L957F) in addition to the N408D mutation that has been associated with ganciclovir and cidofovir resistance. Long-term exposure of HCMV to either ganciclovir or foscarnet ultimately resulted in the selection of multiple UL54 mutations that conferred high levels of resistance to all approved HCMV DNA polymerase inhibitors, i.e., ganciclovir, cidofovir, and foscarnet. Emergence of each viral mutation conferred stepwise increases in drug 50% inhibitory concentrations that could be objectively measured with the new reporter cell assay.     

In vitro resistance profile of the human immunodeficiency virus type 1 protease inhibitor BMS-232632

BMS-232632 is an azapeptide human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor that displays potent anti-HIV-1 activity (50% effective concentration [EC(50)], 2.6 to 5.3 nM; EC(90), 9 to 15 nM). In vitro passage of HIV-1 RF in the presence of inhibitors showed that BMS-232632 selected for resistant variants more slowly than nelfinavir or ritonavir did. Genotypic and phenotypic analysis of three different HIV strains resistant to BMS-232632 indicated that an N88S substitution in the viral protease appeared first during the selection process in two of the three strains. An I84V change appeared to be an important substitution in the third strain used. Mutations were also observed at the protease cleavage sites following drug selection. The evolution to resistance seemed distinct for each of the three strains used, suggesting multiple pathways to resistance and the importance of the viral genetic background. A cross-resistance study involving five other protease inhibitors indicated that BMS-232632-resistant virus remained sensitive to saquinavir, while it showed various levels (0. 1- to 71-fold decrease in sensitivity)-of cross-resistance to nelfinavir, indinavir, ritonavir, and amprenavir. In reciprocal experiments, the BMS-232632 susceptibility of HIV-1 variants selected in the presence of each of the other HIV-1 protease inhibitors showed that the nelfinavir-, saquinavir-, and amprenavir-resistant strains of HIV-1 remained sensitive to BMS-232632, while indinavir- and ritonavir-resistant viruses displayed six- to ninefold changes in BMS-232632 sensitivity. Taken together, our data suggest that BMS-232632 may be a valuable protease inhibitor for use in combination therapy.     

Natural polymorphisms in the human immunodeficiency virus type 2 protease can accelerate time to development of resistance to protease inhibitors

Human immunodeficiency virus type 2 (HIV-2) contains numerous natural polymorphisms in its protease (PR) gene that are implicated in drug resistance in the case of HIV-1. This study evaluated emergent PR resistance in HIV-2. Three HIV-2 isolates were selected for resistance to amprenavir (APV), nelfinavir (NFV), indinavir (IDV), and tipranavir (TPV) in cell culture. Genotypic analysis determined the time to the appearance of protease inhibitor (PI)-associated mutations compared to HIV-1. Phenotypic drug susceptibility assays were used to determine the levels of drug resistance. Within 10 to 15 weeks of serial passage, three major mutations--I54M, I82F, and L90M--arose in HIV-2 viral cultures exposed to APV, NFV, and IDV, whereas I82L was selected with TPV. After 25 weeks, other cultures had developed I50V and I84V mutations. In contrast, no major PI mutations were selected in HIV-1 over this period except for D30N in the context of NFV selective pressure. The baseline phenotypes of wild-type HIV-2 isolates were in the range observed for HIV-1, except for APV and NFV for which a lower degree of sensitivity was seen. The acquisition of the I54M, I84V, L90M, and L99F mutations resulted in multi-PI-resistant viruses, conferring 10-fold to more than 100-fold resistance. Of note, we observed a 62A/99F mutational motif that conferred high-level resistance to PIs, as well as novel secondary mutations, including 6F, 12A, and 21K. Thus, natural polymorphisms in HIV-2 may facilitate the selection of PI resistance. The increasing incidence of such polymorphisms in drug-naive HIV-1- and HIV-2-infected persons is of concern.     

In Vitro Phenotypic Characterization of Hepatitis C Virus NS3 Protease Variants Observed in Clinical Studies of Telaprevir

Telaprevir is a linear, peptidomimetic small molecule that inhibits hepatitis C virus (HCV) replication by specifically inhibiting the NS3·4A protease. In phase 3 clinical studies, telaprevir in combination with peginterferon and ribavirin (PR) significantly improved sustained virologic response (SVR) rates in genotype 1 chronic HCV-infected patients compared with PR alone. In patients who do not achieve SVR after treatment with telaprevir-based regimens, variants with mutations in the NS3·4A protease region have been observed. Such variants can contribute to drug resistance and limit the efficacy of treatment. To gain a better understanding of the viral resistance profile, we conducted phenotypic characterization of the variants using HCV replicons carrying site-directed mutations. The most frequently observed (significantly enriched) telaprevir-resistant variants, V36A/M, T54A/S, R155K/T, and A156S, conferred lower-level resistance (3- to 25-fold), whereas A156T and V36M+R155K conferred higher-level resistance (>25-fold) to telaprevir. Rarely observed (not significantly enriched) variants included V36I/L and I132V, which did not confer resistance to telaprevir; V36C/G, R155G/I/M/S, V36A+T54A, V36L+R155K, T54S+R155K, and R155T+D168N, which conferred lower-level resistance to telaprevir; and A156F/N/V, V36A+R155K/T, V36M+R155T, V36A/M+A156T, T54A+A156S, T54S+A156S/T, and V36M+T54S+R155K, which conferred higher-level resistance to telaprevir. All telaprevir-resistant variants remained fully sensitive to alpha interferon, ribavirin, and HCV NS5B nucleoside and nonnucleoside polymerase inhibitors. In general, the replication capacity of telaprevir-resistant variants was lower than that of the wild-type replicon.     

Novel human immunodeficiency virus type 1 protease mutations potentially involved in resistance to protease inhibitors

Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naive patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 A of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naive patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.     

HIV-1 genotype resistance pattern and evolution in patients failing nelfinavir-containing regimens

Clinical and in vitro studies have suggested that nelfinavir (NFV)-containing regimens may not preclude the use of other protease inhibitors (PIs) in treatment sequencing. We have studied the prevalence of 30N mutation in a human immunodeficiency virus-1 (HIV-1)-infected cohort and the virological response to a PI-containing regimen in patients who had previously failed NFV. A total of 335 patients were included in the study; 32 of them were antiretroviral-naive and 303 were antiretroviral-experienced (251 were PI-experienced). Mutations 30N and/or 90M were not detected in sequences obtained either from the antiretroviral naive or non-PI-experienced patients. The 30N mutation was detected in 21/251 (8.3%) of PI-experienced patients and 90M in 103/251 (41%). Moreover, we have observed that the 88D and 77I mutations were present in more than 75% of patients harbouring the 30N HIV-1 variant and the 71T mutation was present in almost 50% of them. Finally, mutations 30N+90M were never detected together in the same HIV-1 strain. The 30N and 90M mutations were not observed together. The presence of mutations at positions 36, 46, 71, 77, and/or 88 in a 30N background, increases the risk of the cross-resistance to other PIs. The use of NFV as a first-line PI, as an application of drug sequencing strategies, may help preserve future PI options.