Sequence analysis of an HIV-1 isolate which displays unusually high-level AZT resistance in vitro

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01075-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 491-501 ◽

Cited By ~ 51

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01183-10 ◽

2011 ◽

Vol 55 (4) ◽

pp. 1366-1376 ◽

Cited By ~ 22

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.

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Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

Journal of Virology ◽

10.1128/jvi.02203-06 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7852-7859 ◽

Cited By ~ 57

Author(s):

Jessica H. Brehm ◽

Dianna Koontz ◽

Jeffrey D. Meteer ◽

Vinay Pathak ◽

Nicolas Sluis-Cremer ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Rnase H ◽

Cross Resistance ◽

Polymerase Domain ◽

Immunodeficiency Virus ◽

Azt Resistance ◽

Hiv 1

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

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Sustained IFN-I stimulation impairs MAIT cell responses to bacteria by inducing IL-10 during chronic HIV-1 infection

Science Advances ◽

10.1126/sciadv.aaz0374 ◽

2020 ◽

Vol 6 (8) ◽

pp. eaaz0374 ◽

Cited By ~ 4

Author(s):

X. Tang ◽

S. Zhang ◽

Q. Peng ◽

L. Ling ◽

H. Shi ◽

...

Keyword(s):

Cell Activation ◽

Surface Protein ◽

Interleukin 10 ◽

Mait Cells ◽

Cell Dysfunction ◽

Cell Responses ◽

High Level ◽

Hiv 1 ◽

Mait Cell

Mucosal-associated invariant T (MAIT) cells in HIV-1–infected individuals are functionally impaired by poorly understood mechanisms. Single-cell transcriptional and surface protein analyses revealed that peripheral MAIT cells from HIV-1–infected subjects were highly activated with the up-regulation of interferon (IFN)–stimulated genes as compared to healthy individuals. Sustained IFN-α treatment suppressed MAIT cell responses to Escherichia coli by triggering high-level interleukin-10 (IL-10) production by monocytes, which subsequently inhibited the secretion of IL-12, a crucial costimulatory cytokine for MAIT cell activation. Blocking IFN-α or IL-10 receptors prevented MAIT cell dysfunction induced by HIV-1 exposure in vitro. Moreover, blocking the IL-10 receptor significantly improved anti–Mycobacterium tuberculosis responses of MAIT cells from HIV-1–infected patients. Our findings demonstrate the central role of the IFN-I/IL-10 axis in MAIT cell dysfunction during HIV-1 infection, which has implications for the development of anti–IFN-I/IL-10 strategies against bacterial coinfections in HIV-1–infected patients.

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Transmitted Human Immunodeficiency Virus Type 1 Carrying the D67N or K219Q/E Mutation Evolves Rapidly to Zidovudine Resistance In Vitro and Shows a High Replicative Fitness in the Presence of Zidovudine

Journal of Virology ◽

10.1128/jvi.78.14.7545-7552.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7545-7552 ◽

Cited By ~ 31

Author(s):

J. Gerardo García-Lerma ◽

Hamish MacInnes ◽

Diane Bennett ◽

Hillard Weinstock ◽

Walid Heneine

Keyword(s):

Resistance Mutations ◽

Immunodeficiency Virus ◽

Evolution Of Resistance ◽

Drug Naïve ◽

Azt Resistance ◽

The Impact ◽

Hiv 1 ◽

Selection Of

ABSTRACT Drug-naive patients infected with drug-resistant human immunodeficiency virus type 1 (HIV-1) who initiate antiretroviral therapy show a shorter time to virologic failure than patients infected with wild-type (WT) viruses. Resistance-related HIV genotypes not commonly seen in treated patients, which likely result from reversion or loss of primary resistance mutations, have also been recognized in drug-naive persons. Little work has been done to characterize the patterns of mutations in these viruses and the frequency of occurrence, their association with phenotypic resistance, and their effect on fitness and evolution of resistance. Through the analysis of resistance mutations in 1082 newly diagnosed antiretroviral-naive persons from the United States, we found that 35 of 48 (72.9%) persons infected with HIV-1 containing thymidine analog mutations (TAMs) had viruses that lacked a primary mutation (T215Y/F, K70R, or Q151M). Of these viruses, 9 (25.7%) had only secondary TAMs (D67N, K219Q, M41L, or F77L), and all were found to be sensitive to zidovudine (AZT) and other drugs. To assess the impact of secondary TAMs on the evolution of AZT resistance, we generated recombinant viruses from cloned plasma-derived reverse transcriptase sequences. Two viruses had D67N, three had D67N and K219Q/E, and three were WT. Four site-directed mutants with D67N, K219Q, K219E, and D67N/K219Q were also made in HIV-1HXB2. In vitro selection of AZT resistance showed that viruses with D67N and/or K219Q/E acquired AZT resistance mutations more rapidly than WT viruses (36 days compared to 54 days; P = 0.003). To investigate the factors associated with the rapid selection of AZT mutations in these viruses, we evaluated fitness differences among HXB2WT and HXB2D67N or HXB2D67N/K219Q in the presence of AZT. Both HXB2D67N/K219Q and HXB2D67N were more fit than HXB2WT in the presence of either low or high AZT concentrations, likely reflecting low-level resistance to AZT that is not detectable by phenotypic testing. In the absence of AZT, the fitness cost conferred by D67N or K219Q was modest. Our results demonstrate that viruses with unique patterns of TAMs, including D67N and/or K219Q/E, are commonly found among newly diagnosed persons and illustrate the expanding diversity of revertant viruses in this population. The modest fitness cost conferred by D67N and K219Q supports persistence of these mutants in the untreated population and highlights the potential for secondary transmission. The faster evolution of these mutants toward AZT resistance is consistent with the higher viral fitness in the presence of AZT and shows that these viruses are phenotypically different from WT HIV-1. Our study emphasizes the need for clinical studies to better define the impact of these mutants on treatment responses and evolution of resistance.

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Drug Combination of AZT and ddl: Synergism of Action and Prevention of Appearance of AZT-Resistance

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029400500108 ◽

1994 ◽

Vol 5 (1) ◽

pp. 51-55 ◽

Cited By ~ 6

Author(s):

G. Antonelli ◽

F. Dianzani ◽

D. Bellarosa ◽

O. Turriziani ◽

E. Riva ◽

...

Keyword(s):

In Vitro Selection ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Immunodeficiency Virus ◽

Resistant Strains ◽

Azt Resistance ◽

Hiv 1

Both 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxynosine (ddl) strongly inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Here, it is shown that combination of AZT and ddl at concentrations that are readily achievable in vivo synergistically inhibit HIV-1 replication in C8166 cells and peripheral blood mononuclear cells. The synergism is significant even when the effect of AZT and ddl alone was negligible. Our findings show that AZT-resistance is less likely to occur when a combination of AZT and ddl is used. Particularly, generation of AZT-resistant strains by in vitro selection is prevented, or delayed, by the combination of AZT plus ddl. Taken together these observations provide a rationale for combination of AZT and ddl in the therapy of AIDS patients.

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Immunoglobulin G (IgG) and IgA, but also Nonantibody Factors, Account for In Vitro Neutralization of Human Immunodeficiency Virus (HIV) Type 1 Primary Isolates by Serum and Plasma of HIV-Infected Patients

Journal of Virology ◽

10.1128/jvi.75.11.5421-5424.2001 ◽

2001 ◽

Vol 75 (11) ◽

pp. 5421-5424 ◽

Cited By ~ 27

Author(s):

Renaud Burrer ◽

Dominique Salmon-Ceron ◽

Sophie Richert ◽

Gianfranco Pancino ◽

Gabriella Spiridon ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Broad Spectrum ◽

Plasma Samples ◽

Two Samples ◽

Immunodeficiency Virus ◽

Hiv Type 1 ◽

High Level ◽

Hiv 1

ABSTRACT The factors present in serum and plasma samples of human immunodeficiency virus (HIV)-infected patients that are responsible for the neutralization of four HIV type 1 (HIV-1) primary isolates in vitro have been analyzed. Purification of immunoglobulins (Ig) by affinity chromatography showed that the activities were mostly attributable to IgG and less frequently to IgA. For two samples, we have shown that the high-level and broad-spectrum inhibitory activity was essentially caused by non-Ig factors interfering with the measurement of antibody-specific neutralizing activity.

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In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa401 ◽

2020 ◽

Vol 76 (1) ◽

pp. 130-134

Author(s):

Francesco Saladini ◽

Federica Giammarino ◽

Behnaz A Hosseini ◽

Alessia Giannini ◽

Adele Boccuto ◽

...

Keyword(s):

In Vitro Study ◽

Cross Resistance ◽

Reference List ◽

Resistance Mutations ◽

Nnrti Resistance ◽

Infectious Clones ◽

High Level ◽

Reduced Activity ◽

Hiv 1

Abstract Objectives Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. Methods In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. Results Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were >10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P < 0.001). Conclusions Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

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Lactobacilli Expressing Broadly Neutralizing Nanobodies against HIV-1 as Potential Vectors for HIV-1 Prophylaxis?

Vaccines ◽

10.3390/vaccines8040758 ◽

2020 ◽

Vol 8 (4) ◽

pp. 758

Author(s):

Sarah Kalusche ◽

Kanika Vanshylla ◽

Franziska Kleipass ◽

Henning Gruell ◽

Barbara Müller ◽

...

Keyword(s):

High Risk ◽

Neutralizing Antibodies ◽

Sexual Transmission ◽

Lactobacillus Rhamnosus ◽

Passive Immunization ◽

Acidic Ph ◽

Human Vagina ◽

High Level ◽

Hiv 1

In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.

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