Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.     

Lopinavir Plasma Concentrations and Virological Outcome with Lopinavir-Ritonavir Monotherapy in HIV-1-Infected Patients

There is significant intra- and intersubject variability in lopinavir (LPV) plasma concentrations after standard dosing; thus, this prospective study was conducted to determine whether low plasma LPV concentrations could be associated with virological outcome throughout lopinavir-ritonavir maintenance monotherapy (mtLPVr) in the clinical practice setting. If this hypothesis would be confirmed, LPV drug monitoring could improve the efficacy of mtLPVr regimens. Patients with previous virological failure (VF) on protease inhibitor-based regimens were also included if the genotypic resistance tests showed no major resistance mutation associated with reduced susceptibility to lopinavir-ritonavir. VF was defined as 2 consecutive determinations of HIV RNA levels of >200 copies/ml. Efficacy was analyzed by per-protocol analysis. Plasma LPV trough concentrations were measured by high-performance liquid chromatography using a UV detector. A total of 127 patients were included (22% with previous failure on protease inhibitors). After 96 weeks, the efficacy rate was 82.3% (95% confidence interval [CI₉₅], 75.3 to 89.3%). Virological efficacy was independent of LPV plasma concentrations even when LPVr was given once daily. An adherence of <90% (HR, 4.4 [CI₉₅, 1.78 to 10.8; P = 0.001]) and the presence of blips in the preceding 12 months (HR, 3.06 [CI₉₅, 1.17 to 8.01; P = 0.022]) were the only variables independently associated with time to VF. These findings suggest that the LPV concentrations achieved with the standard doses of LPVr are sufficient to maintain virological control during monotherapy and that measurement of LPV concentrations is not useful for predicting virological outcome. Tight control of viral replication in the previous months and strict adherence throughout the mtLPVr regimen could improve the virological efficacy of this maintenance regimen.     

Prevalent Polymorphisms in Wild-Type HIV-1 Integrase Are Unlikely To Engender Drug Resistance to Dolutegravir (S/GSK1349572)

The majority of HIV-1 integrase amino acid sites are highly conserved, suggesting that most are necessary to carry out the critical structural and functional roles of integrase. We analyzed the 34 most variable sites in integrase (>10% variability) and showed that prevalent polymorphic amino acids at these positions did not affect susceptibility to the integrase inhibitor dolutegravir (S/GSK1349572), as demonstrated both in vitro (in site-directed mutagenesis studies) and in vivo (in a phase IIa study of dolutegravir monotherapy in HIV-infected individuals). Ongoing clinical trials will provide additional data on the virologic activity of dolutegravir across subject viruses with and without prevalent polymorphic substitutions.     

浙江省丽水市男男性行为人群HIV-1基因亚型和传播特征研究

目的了解浙江省丽水市男男性行为人群（MSM）人类免疫缺陷病毒1型（HIV-1）基因亚型和传播特征。方法收集丽水市2015－2018年新确证且未经抗病毒治疗MSM HIV-1感染样本,扩增pol基因并测序,构建系统进化树分析基因亚型及成簇特征,通过CPR 6.0在线软件分析耐药情况。结果105例样本成功获得89例合格序列,优势亚型为CRF01_AE（49.4%,44/89）和CRF07_BC（37.1%,33/89）,其他包括B和CRF55_01B各3例,CRF08_BC、CRF59_01B和独特重组型 （01_AE/BC）各2例。 发现8个传播簇,总成簇比例为21.3%（19/89）,其中7个传播簇与现住址为丽水市莲都区的病例有关（87.5%,7/8）。 发现3例样本存在1个监测性耐药突变,分别为M46I、V106A、K103N,传播性耐药率为3.4%（3/89）。结论丽水市7种MSM HIV-1亚型共存,呈现多元化趋势,莲都区发挥关键传播作用,应加强监测并重点对传播簇病例开展靶向干预。 耐药传播率处于低流行水平。     

Genotypic and Phenotypic Characterization of Human Immunodeficiency Virus Type 1 Variants Isolated from Patients Treated with the Protease Inhibitor Nelfinavir

Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.     

Atazanavir trough plasma concentration monitoring in a cohort of HIV-1-positive individuals receiving highly active antiretroviral therapy

OBJECTIVES: Atazanavir is a recently approved HIV protease inhibitor (PI). As with other PIs, careful attention to potential pharmacokinetic drug interactions in clinical practice is necessary. The aim of this study was to assess the clinical associations with plasma atazanavir concentrations in HIV-positive individuals. METHODS: Individuals established on an atazanavir-containing regimen, completed an interviewer-administered questionnaire recording atazanavir dosing characteristics, concomitant medication use and adherence. After completion, plasma atazanavir concentrations were measured. RESULTS: Of 100 individuals, mean trough plasma atazanavir concentrations (mug/L) were 282 (95% CI 95-468, n = 19) and 774 (95% CI 646-902, n = 81) in those on non- and ritonavir-boosted atazanavir regimens, respectively. Eighty-five individuals had HIV RNA <50 copies/mL. Seven individuals had atazanavir plasma concentrations below the assay limit of detection (<50 microg/L), all of whom had undetectable plasma HIV RNA. In a multivariate analysis, nevirapine use was associated with significantly lower trough atazanavir concentrations (P = 0.011) and lopinavir/ritonavir use with higher trough atazanavir concentrations (P = 0.032). Dosing characteristics (including food taken), concomitant medications (including drugs used for dyspepsia) and HIV RNA were not significantly associated with trough atazanavir concentrations. CONCLUSIONS: In this cohort, despite the wide inter-individual variability of atazanavir trough concentrations, no significant association with dosing characteristics, concomitant medication (with the exception of nevirapine and lopinavir/ritonavir) or virological response was observed. Further work is needed to assess the optimal dosing regimen when using atazanavir with nevirapine.     

Pharmacokinetic Interactions between BMS-626529, the Active Moiety of the HIV-1 Attachment Inhibitor Prodrug BMS-663068, and Ritonavir or Ritonavir-Boosted Atazanavir in Healthy Subjects

BMS-663068 is a prodrug of BMS-626529, a first-in-class attachment inhibitor that binds directly to HIV-1 gp120, preventing initial viral attachment and entry into host CD4⁺ T cells. This open-label, multiple-dose, four-sequence, crossover study addressed potential two-way drug-drug interactions following coadministration of BMS-663068 (BMS-626529 is a CYP3A4 substrate), atazanavir (ATV), and ritonavir (RTV) (ATV and RTV are CYP3A4 inhibitors). Thirty-six healthy subjects were randomized 1:1:1:1 to receive one of four treatment sequences with three consecutive treatments: BMS-663068 at 600 mg twice daily (BID), BMS-663068 at 600 mg BID plus RTV at 100 mg once daily (QD), ATV at 300 mg QD plus RTV at 100 mg QD (RTV-boosted ATV [ATV/r]), or BMS-663068 at 600 mg BID plus ATV at 300 mg QD plus RTV at 100 mg QD. Compared with the results obtained by administration of BMS-663068 alone, coadministration of BMS-663068 with ATV/r increased the BMS-626529 maximum concentration in plasma (C_max) and the area under the concentration-time curve in one dosing interval (AUC_tau) by 68% and 54%, respectively. Similarly, coadministration of BMS-663068 with RTV increased the BMS-626529 C_max and AUC_tau by 53% and 45%, respectively. Compared with the results obtained by administration of ATV/r alone, ATV and RTV systemic exposures remained similar following coadministration of BMS-663068 with ATV/r. BMS-663068 was generally well tolerated, and there were no adverse events (AEs) leading to discontinuation, serious AEs, or deaths. Moderate increases in BMS-626529 systemic exposure were observed following coadministration of BMS-663068 with ATV/r or RTV. However, the addition of ATV to BMS-663068 plus RTV did not further increase BMS-626529 systemic exposure. ATV and RTV exposures remained similar following coadministration of BMS-663068 with either ATV/r or RTV. BMS-663068 was generally well tolerated alone or in combination with either RTV or ATV/r.     

Role of P-Glycoprotein in the Distribution of the HIV Protease Inhibitor Atazanavir in the Brain and Male Genital Tract

The blood-testis barrier and blood-brain barrier are responsible for protecting the male genital tract and central nervous system from xenobiotic exposure. In HIV-infected patients, low concentrations of antiretroviral drugs in cerebrospinal fluid and seminal fluid have been reported. One mechanism that may contribute to reduced concentrations is the expression of ATP-binding cassette drug efflux transporters, such as P-glycoprotein (P-gp). The objective of this study was to investigate in vivo the tissue distribution of the HIV protease inhibitor atazanavir in wild-type (WT) mice, P-gp/breast cancer resistance protein (Bcrp)-knockout (Mdr1a^−/−, Mdr1b^−/−, and Abcg2^−/− triple-knockout [TKO]) mice, and Cyp3a^−/− (Cyp) mice. WT mice and Cyp mice were pretreated with a P-gp/Bcrp inhibitor, elacridar (5 mg/kg of body weight), and the HIV protease inhibitor and boosting agent ritonavir (2 mg/kg intravenously [i.v.]), respectively. Atazanavir (10 mg/kg) was administered i.v. Atazanavir concentrations in plasma (C_plasma), brain (C_brain), and testes (C_testes) were quantified at various times by liquid chromatography-tandem mass spectrometry. In TKO mice, we demonstrated a significant increase in atazanavir C_brain/C_plasma (5.4-fold) and C_testes/C_plasma (4.6-fold) ratios compared to those in WT mice (P < 0.05). Elacridar-treated WT mice showed a significant increase in atazanavir C_brain/C_plasma (12.3-fold) and C_testes/C_plasma (13.5-fold) ratios compared to those in vehicle-treated WT mice. In Cyp mice pretreated with ritonavir, significant (P < 0.05) increases in atazanavir C_brain/C_plasma (1.8-fold) and C_testes/C_plasma (9.5-fold) ratios compared to those in vehicle-treated WT mice were observed. These data suggest that drug efflux transporters, i.e., P-gp, are involved in limiting the ability of atazanavir to permeate the rodent brain and genital tract. Since these transporters are known to be expressed in humans, they could contribute to the low cerebrospinal and seminal fluid antiretroviral concentrations reported in the clinic.     

Resistance profiles of cyclic and linear inhibitors of HIV-1 protease

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.     

Characterization of V36C,a Novel Amino Acid Substitution Conferring Hepatitis C Virus (HCV) Resistance to Telaprevir,a Potent Peptidomimetic Inhibitor of HCV Protease

We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.Advances in virology have led to the development of novel therapeutics specifically targeting hepatitis C virus (HCV) (4). Telaprevir (VX-950; Vertex Pharmaceuticals Incorporated, Cambridge, MA) is a novel, highly selective, potent peptidomimetic inhibitor of the HCV nonstructural protein 3/4A (NS3/4A) protease (1, 3) which has reached phase III clinical development in combination with pegylated alpha interferon (IFN-α) and ribavirin. Amino acid substitutions conferring telaprevir resistance have been reported at positions Val 36, Thr 54, Arg 155, and Ala 156 of the NS3 protease (2, 5). In patients treated with telaprevir and pegylated IFN-α with and without ribavirin, breakthroughs during treatment and relapses after treatment are characterized by the recurrence of telaprevir-resistant HCV variant replication (1).Here, we characterized a novel, so far unknown, telaprevir resistance substitution at position Val 36 in a 38-year-old treatment-naïve woman with chronic hepatitis C due to HCV genotype 1b infection. The patient was enrolled in PROVE2, a phase II randomized clinical trial assessing the efficacy and safety of telaprevir in combination with pegylated IFN-α2a with or without ribavirin (1). The patient was treated with telaprevir at 750 mg/8 h, pegylated IFN-α2a at 180 μg/week, and ribavirin at 1.0 g/day. HCV RNA became undetectable (<10 IU/ml) on therapy, but after 43 days of treatment, the patient withdrew consent and stopped therapy. She continued to be followed up after treatment withdrawal.Figure ​Figure11 shows the kinetics of HCV RNA levels in the patient during the 43 days of therapy. HCV RNA was detected 8 weeks after treatment withdrawal, and HCV RNA levels returned to nearly baseline levels. Twenty to 24 full-length NS3 protease clones were sequenced at each HCV RNA-positive time point. The patient was infected with a wild-type, telaprevir sensitive viral population at the baseline (Fig. ​(Fig.1).1). At the time of posttreatment relapse, the HCV variants all bore a Val-to-Cys substitution at position 36 (V36C). The V36C substitution remained dominant throughout posttreatment follow-up, up to day 512 after the start of therapy (Fig. ​(Fig.1).1). This substitution was associated with a Leu-to-Phe substitution at position 14 of the protease (L14F). L14F is present in approximately 6% of all HCV sequences and 8.5% of HCV subtype 1b sequences available in the European HCV database (http://euhcvdb.ibcp.fr/euHCVdb/). Based on molecular modeling, it is predicted to be located at more than 20 Å of the telaprevir binding site and is therefore unlikely to alter telaprevir-NS3 protease interaction.Open in a separate windowFIG. 1.(Top) HCV RNA kinetics in a patient who selected a V36C amino acid substitution on triple therapy with pegylated IFN-α2a, ribavirin, and telaprevir. The HCV RNA levels are expressed in log₁₀ IU/ml. LLD, lower limit of detection. The circled dots represent the four time points at which quasispecies sequence analysis was performed. (Bottom) Dynamics of NS3 quasispecies populations after treatment withdrawal relative to the baseline.The V36C substitution was further characterized and compared to other substitutions at NS3 protease position 36 observed in dominant populations in the PROVE2 trial (Table ​(Table1).1). The methods are described in the supplemental material. As shown in Table ​Table1,1, the V36C substitution conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration (IC₅₀) in our NS3/4A protease enzyme assay. This moderate increase in the IC₅₀ was on the same order as that conferred by V36M and slightly greater than that conferred by V36L. The K_m and V_max values of the NS3 protease-catalyzed enzymatic reaction were on the same order for the V36C variant and the other V36 variants and close to that of the wild-type protease (Table ​(Table11).

TABLE 1.

V36 substitutions observed in patients included in the PROVE2 clinical trial