Variability of the HIV-1 3′ polypurine tract (3′PPT) region and implication in integrase inhibitor resistance

2019 ◽  
Vol 74 (12) ◽  
pp. 3440-3444 ◽  
Author(s):  
Isabelle Malet ◽  
Olivier Delelis ◽  
Thuy Nguyen ◽  
Valentin Leducq ◽  
Besma Abdi ◽  
...  
Keyword(s):  
Rapid Change ◽  
Rare Event ◽  
Integration Step ◽  
Integrase Gene ◽  
Polypurine Tract ◽  
Treatment Naïve ◽  
High Level ◽  
Hiv 1 ◽  
Level Resistance

Abstract Background Integrase strand-transfer inhibitors (INSTIs) are efficient at impairing retroviral integration, which is a critical step in HIV-1 replication. To date, resistance to these compounds has been explained by mutations in the viral protein integrase, which catalyses the integration step. Recently, it has been shown that selected mutations in the 3′ polypurine tract (3′PPT), a sequence involved in the reverse transcription mechanism, result in high-level resistance to these compounds. This observation was reinforced by the description of a patient who failed INSTI treatment by selecting mutations in the 3′PPT sequence. Methods Sequences of the 3′PPT region were analysed in 30706 treatment-naive patients from the public Los Alamos database belonging to six different subtypes and, in parallel, in 107 patients failing INSTI treatment. Results The analysis showed that the sequences of patients failing INSTI treatment, in the same way as those of treatment-naive patients, are very well conserved regardless of the presence or absence of resistance mutations in the integrase gene. Conclusions This study confirms that the selection of a mutation in the 3′PPT region conferring high-level resistance to INSTIs is a rare event. It would require a particular in vivo context and especially a long enough time to be selected, this exposure time being generally reduced by the rapid change of treatment in the case of virological failure. Larger-scale studies in patients with INSTI treatment failure are needed to determine whether the 3′PPT region can play an important role in vivo in INSTI resistance.

scholarly journals In VitroCharacterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile

2011 ◽  
Vol 55 (4) ◽  
pp. 1366-1376 ◽  
Author(s):  
Christian Callebaut ◽  
Kirsten Stray ◽  
Luong Tsai ◽  
Matt Williams ◽  
Zheng-Yu Yang ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Cell Types ◽  
Human Serum Protein ◽  
Treatment Naïve ◽  
Low Cytotoxicity ◽  
T Cell Lines ◽  
High Level ◽  
Hiv 1

ABSTRACTGS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to theparaposition of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki= 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50= 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs,in vitrohepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boostingin vivo. In summary, results from this broadin vitropharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

scholarly journals Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

2019 ◽  
Author(s):  
Ean Spielvogel ◽  
Sook-Kyung Lee ◽  
Shuntai Zhou ◽  
Gordon J. Lockbaum ◽  
Mina Henes ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Selective Pressure ◽  
Viral Fitness ◽  
Fourth Generation ◽  
Genetic Barrier ◽  
High Level ◽  
Hiv 1 ◽  
Level Resistance ◽  
Selection Of

SummaryWell-designed viral protease inhibitors (PIs) potently inhibit replication as well as create a high genetic barrier for resistance. Through in vivo selective pressure, we have generated high-level resistance against ten HIV-1 PIs and their precursor, the FDA-approved drug darunavir (DRV), achieving 1,000-fold resistance over the starting EC50. The accumulation of mutations revealed two pathways to high-level resistance, resulting in protease variants with up to 14 mutations in and outside of the active site. The two pathways demonstrate the interplay between drug resistance and viral fitness. Replicate selections showed that one inhibitor could select for resistance through either pathway, although subtle changes in chemical structure of the inhibitors led to preferential use of one pathway over the other. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high-level of selective pressure that is attainable with these fourth-generation protease inhibitors, and the interplay between selection of mutations to confer resistance while maintaining viral fitness.

scholarly journals Drug Resistance-Associated Mutations in Antiretroviral Treatment-Experienced Patients in Kuwait

2018 ◽  
Vol 27 (2) ◽  
pp. 152-157
Author(s):  
Wassim Chehadeh ◽  
Osama Albaksami ◽  
Sonia Elezebeth John ◽  
Widad Al-Nakib
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Antiretroviral Drugs ◽  
First Line ◽  
Genotypic Resistance ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
High Level ◽  
Interpretation Algorithm ◽  
Hiv 1 ◽  
Level Resistance

Objectives: To investigate the prevalence of nonpolymorphic resistance-associated mutations (RAM) in HIV-1 patients on first-line antiretroviral therapy in Kuwait. Subjects and Methods: Total RNA was isolated from plasma samples of 42 patients who received a first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. HIV-1 protease and reverse transcriptase genetic regions were then amplified by nested reverse transcription-polymerase chain reaction and directly sequenced. The HIV-1 subtype was identified using the Bayesian phylogenetic method, and RAM were identified using the Stanford University genotypic resistance interpretation algorithm. Results: The HIV-1 viral load at sampling ranged from < 20 to 8.25 × 104 copies/ml. CRF01_AE, C, and B were the most predominant HIV-1 subtypes. Nonpolymorphic mutations associated with resistance to antiretroviral drugs were detected in 11 (26.2%) of the 42 patients; 5 (11.9%) patients had mutations associated with a high-level resistance to nucleoside reverse transcriptase inhibitors (NRTI), 4 (9.5%) patients had mutations associated with resistance to NNRTI, 1 (2.4%) patient had mutations associated with resistance to both NRTI and NNRTI, and 1 (2.4%) patient had mutations potentially associated with low-level resistance to both protease inhibitors and NNRTI. All patients with RAM had a detectable plasma HIV-1 RNA level. Conclusion: Our results indicate the development of RAM during an NNRTI-based regimen and highlight the importance of considering other regimens to avoid treatment failure.

Rapid Emergence of Daptomycin Resistance in Nonstriatum Corynebacterium Species: A Multicenter Study

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S32-S33
Author(s):  
Kaitlin Mitchell ◽  
Erin McElvania ◽  
Meghan Wallace ◽  
Amy Robertson ◽  
Lars Westblade ◽  
...  
Keyword(s):  
Multicenter Study ◽  
Tertiary Care ◽  
Total N ◽  
Gradient Diffusion ◽  
Corynebacterium Species ◽  
Short Period ◽  
High Level ◽  
Level Resistance

Abstract Members of the genus Corynebacterium are increasingly recognized as causes of opportunistic infection; some species can be multidrug resistant, posing a treatment challenge. Daptomycin is frequently used as therapy of last resort in this setting, but previous work from our group demonstrated the ability of C striatum clinical isolates to rapidly develop high-level resistance to daptomycin, both in vivo and in vitro. Here, our objective was to expand this investigation into a multicenter study evaluating multiple Corynebacterium species. Corynebacterium strains from three tertiary-care academic medical centers (total, n = 76; site 1, n = 44; site 2, n = 15; site 3, n = 17) were evaluated, representing 16 species. Isolates were identified during routine clinical testing and reported to species level in accordance with each laboratory’s standard operating procedures. Identification of each species was confirmed using both VITEK MS and Bruker BioTyper MALDI-TOF MS. MICs to daptomycin (Etest), vancomycin (Etest), and telavancin (Liofilchem) at baseline were determined using gradient diffusion methods on Mueller-Hinton agar with blood (Hardy Diagnostics). Each isolate was then inoculated in duplicate to 5 mL Tryptic Soy Broth. A daptomycin Etest was submerged in one tube from each pair, and growth was observed after 24-hour incubation. If turbidity was observed in the tube with daptomycin, MICs for each of the 3 antimicrobials were reassessed. High-level daptomycin resistance emerged in 24 strains: C aurimucosum (1/1 isolate tested), C bovis (1/2), C jeikeium (2/11), C macginleyi (3/3), C resistens (1/1), C simulans (1/1), C striatum (14/14 isolates), and C ulcerans (1/1). The majority of these isolates had MIC values >256 µg/mL following exposure to daptomycin. Forty-eight other isolates remained susceptible to daptomycin: C afermentans (1/1), C amycolatum (19/20), C diphtheriae (1/1), C jeikeium (7/11), C kroppenstedtii (2/2), C propinquum (3/3), C pseudodiphtheriticum (6/6), C tuberculostearicum (0/6), and C urealyticum (0/3). Many of these isolates did not undergo MIC testing postdaptomycin exposure in broth due to complete lack of growth. Among those that did (n = 19), the median daptomycin MIC was 0.38 µg/mL (mean 0.42 µg/mL; range 0.023-1.0 µg/mL). One isolate of C bovis and two isolates of C jeikeium yielded variable susceptibility to daptomycin; a subset of resistant colonies grew adjacent to the gradient diffusion strip. Upon isolation and further MIC testing, these colonies maintained high-level resistance. In addition, one isolate of C amycolatum exhibited high-level daptomycin resistance (MIC >256 µg/mL) prior to in vitro exposure. All isolates in the cohort were susceptible to vancomycin and telavancin, both before and after daptomycin exposure. Our findings suggest that multiple Corynebacterium species can rapidly develop high-level daptomycin resistance after a short period of exposure to this antimicrobial. This finding has important clinical implications, especially in the treatment of invasive infections or infections of indwelling medical devices.

scholarly journals Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

2009 ◽  
Vol 54 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Olivier Delelis ◽  
Sylvain Thierry ◽  
Frédéric Subra ◽  
Françoise Simon ◽  
Isabelle Malet ◽  
...  
Keyword(s):  
Viral Genome ◽  
Effective Concentration ◽  
Coding Region ◽  
Delayed Emergence ◽  
Infected Cells ◽  
High Level ◽  
Emergence Of Resistance ◽  
Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

scholarly journals CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 246 ◽  
Author(s):  
Xingliang Wang ◽  
Yanjun Xu ◽  
Jianlei Huang ◽  
Wenzhong Jin ◽  
Yihua Yang ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Deletion Mutation ◽  
Ostrinia Furnacalis ◽  
Corn Borer ◽  
Resistance Evolution ◽  
Loop Region ◽  
Truncating Mutation ◽  
High Level ◽  
Level Resistance

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.

scholarly journals Efficacy of Ampicillin plus Ceftriaxone in Treatment of Experimental Endocarditis Due to Enterococcus faecalis Strains Highly Resistant to Aminoglycosides

1999 ◽  
Vol 43 (3) ◽  
pp. 639-646 ◽  
Author(s):  
Joan Gavaldà ◽  
Carmen Torres ◽  
Carmen Tenorio ◽  
Pedro López ◽  
Myriam Zaragoza ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Enterococcus Faecalis ◽  
Pharmacokinetic Parameters ◽  
Initial Inoculum ◽  
High Level ◽  
Time Kill ◽  
Disk Method ◽  
Level Resistance

The purpose of this work was to evaluate the in vitro possibilities of ampicillin-ceftriaxone combinations for 10 Enterococcus faecalis strains with high-level resistance to aminoglycosides (HLRAg) and to assess the efficacy of ampicillin plus ceftriaxone, both administered with humanlike pharmacokinetics, for the treatment of experimental endocarditis due to HLRAg E. faecalis. A reduction of 1 to 4 dilutions in MICs of ampicillin was obtained when ampicillin was combined with a fixed subinhibitory ceftriaxone concentration of 4 μg/ml. This potentiating effect was also observed by the double disk method with all 10 strains. Time-kill studies performed with 1 and 2 μg of ampicillin alone per ml or in combination with 5, 10, 20, 40, and 60 μg of ceftriaxone per ml showed a ≥2 log10 reduction in CFU per milliliter with respect to ampicillin alone and to the initial inoculum for all 10E. faecalis strains studied. This effect was obtained for seven strains with the combination of 2 μg of ampicillin per ml plus 10 μg of ceftriaxone per ml and for six strains with 5 μg of ceftriaxone per ml. Animals with catheter-induced endocarditis were infected intravenously with 108 CFU of E. faecalis V48 or 105 CFU of E. faecalisV45 and were treated for 3 days with humanlike pharmacokinetics of 2 g of ampicillin every 4 h, alone or combined with 2 g of ceftriaxone every 12 h. The levels in serum and the pharmacokinetic parameters of the humanlike pharmacokinetics of ampicillin or ceftriaxone in rabbits were similar to those found in humans treated with 2 g of ampicillin or ceftriaxone intravenously. Results of the therapy for experimental endocarditis caused by E. faecalis V48 or V45 showed that the residual bacterial titers in aortic valve vegetations were significantly lower in the animals treated with the combinations of ampicillin plus ceftriaxone than in those treated with ampicillin alone (P < 0.001). The combination of ampicillin and ceftriaxone showed in vitro and in vivo synergism against HLRAgE. faecalis.

scholarly journals Effect of drug dose and timing of treatment on the emergence of drug resistance in vivo in a malaria model

2020 ◽  
Vol 2020 (1) ◽  
pp. 196-210 ◽  
Author(s):  
Mónica M Acosta ◽  
Joshua T Bram ◽  
Derek Sim ◽  
Andrew F Read
Keyword(s):  
Drug Resistance ◽  
Drug Treatment ◽  
Drug Dose ◽  
Treatment Timing ◽  
Treatment Regimens ◽  
Resistance Evolution ◽  
Timing Of Treatment ◽  
High Level ◽  
Level Resistance

Abstract Background and objectives There is a significant interest in identifying clinically effective drug treatment regimens that minimize the de novo evolution of antimicrobial resistance in pathogen populations. However, in vivo studies that vary treatment regimens and directly measure drug resistance evolution are rare. Here, we experimentally investigate the role of drug dose and treatment timing on resistance evolution in an animal model. Methodology In a series of experiments, we measured the emergence of atovaquone-resistant mutants of Plasmodium chabaudi in laboratory mice, as a function of dose or timing of treatment (day post-infection) with the antimalarial drug atovaquone. Results The likelihood of high-level resistance emergence increased with atovaquone dose. When varying the timing of treatment, treating either very early or late in infection reduced the risk of resistance. When we varied starting inoculum, resistance was more likely at intermediate inoculum sizes, which correlated with the largest population sizes at time of treatment. Conclusions and implications (i) Higher doses do not always minimize resistance emergence and can promote the emergence of high-level resistance. (ii) Altering treatment timing affects the risk of resistance emergence, likely due to the size of the population at the time of treatment, although we did not test the effect of immunity whose influence may have been important in the case of late treatment. (iii) Finding the ‘right’ dose and ‘right’ time to maximize clinical gains and limit resistance emergence can vary depending on biological context and was non-trivial even in our simplified experiments. Lay summary In a mouse model of malaria, higher drug doses led to increases in drug resistance. The timing of drug treatment also impacted resistance emergence, likely due to the size of the population at the time of treatment.

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