Monolithic anion-exchange chromatography yields rhinovirus of high purity

Allmaier, Günter; Blaas, Dieter; Bliem, Christina; Dechat, Thomas; Fedosyuk, Sofiya; Gösler, Irene; Kowalski, Heinrich; Weiss, Victor U.

doi:10.1016/j.jviromet.2017.09.027

Cited by 10 publications

(5 citation statements)

References 38 publications

(50 reference statements)

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“…In Taiwan, the prevalence rate of genotype 6 was around 28.0% among PWID with chronic HCV/HIV infection 11. Hence, next-generation sequencing may be more suitable to identify mixed infection of HCV before treatment in this population 27. The other patient had an HCV viral load of 4.8 log RNA (68,966 IU/mL and genotype 1b at enrollment), and he achieved undetectable viral loads (<10 IU/mL) at week 4 and week 12 (end of treatment); however, genotype 1b with 3.9 log RNA (2,202 IU/mL) was detectable at week 24 (at follow-up week 12).…”

Section: Discussionmentioning

confidence: 99%

<p>Grazoprevir/elbasvir in peginterferon alfa plus ribavirin experienced patients with chronic genotype 1 HCV/HIV co-infection: a non-randomized, open-label clinical trial</p>

Lin¹,

Li²,

Ku³

et al. 2019

IDR

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Background and aims: We aimed to evaluate the efficacy and tolerability of grazoprevir/elbasvir in patients with chronic genotype 1 hepatitis C virus (HCV) and HIV co-infection who experienced peginterferon alfa plus ribavirin (PegIFN/RBV) (clinicaltrials.gov NCT03098121). Methods: This non-randomized, open-label trial study was conducted in Taoyuan General Hospital. HIV-infected patients were screened for HCV antibody since June 1, 2012, and HCV and HIV co-infected patients were tested for HCV RNA. The subjects who experienced PegIFN/RBV were enrolled in the study, and of whom with chronic genotype 1a or 1b received grazoprevir 100 mg and elbasvir 50 mg in a fixed-dose combination tablet once daily with or without ribavirin for 12 to 16 weeks. Results: Of 2,419 HIV-infected patients, 40 patients with chronic genotype 1 HCV and HIV co-infection who failed PegIFN/RBV treatment were enrolled. Sixteen patients had genotype 1a and 24 patients had genotype 1b, with or without cirrhosis. The median age was 42 (41–47) years, and 5 patients (12.5%) were diagnosed with liver cirrhosis (child Pugh score A). The median CD4 count was 504 cells/μL (321–689). All patients (100%) had HIV viral load <200 copies/mL, and HCV viral load was 6.3 log 10 IU/mL (3.98–7.12). At the end of treatment, all patients (100%, 40/40) had undetectable HCV viral load, and 95.0% (38/40) of patients achieved sustained virologic response at 12 weeks. Conclusion: Grazoprevir/elbasvir was effective in genotype 1 patients co-infected with HIV with or without cirrhosis. This finding is consistent with that of previous trials of this regimen in monoinfected population.

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Section: Discussionmentioning

confidence: 99%

<p>Grazoprevir/elbasvir in peginterferon alfa plus ribavirin experienced patients with chronic genotype 1 HCV/HIV co-infection: a non-randomized, open-label clinical trial</p>

Lin¹,

Li²,

Ku³

et al. 2019

IDR

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“…All experiments were carried out with RV-A2, initially acquired from ATCC and propagated, purified, and polished via monolithic chromatography as described previously ( Allmaier et al, 2018 ). The presence of natural empty particles was evaluated by separating 10 μg of purified RV-A2 on a 15% SDS-polyacrylamide gel; no significant amount of VP0, indicative of natural empties, was detected ( Supplementary Figure S2b ).…”

Section: Methodsmentioning

confidence: 99%

nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

et al. 2020

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Thermal shift assays measure the stability of macromolecules and macromolecular assemblies as a function of temperature. The Particle Stability Thermal Release Assay (PaSTRy) of picornaviruses is based on probes becoming strongly fluorescent upon binding to hydrophobic patches of the protein capsid (e.g., SYPRO Orange) or to the viral RNA genome (e.g., SYTO-82) that become exposed upon heating virus particles. PaSTRy has been exploited for studying the stability of viral mutants, viral uncoating, and the effect of capsid-stabilizing compounds. While the results were usually robust, the thermal shift assay with SYPRO Orange is sensitive to surfactants and EDTA and failed at least to correctly report the effect of excipients on an inactivated poliovirus 3 vaccine. Furthermore, interactions between the probe and capsid-binding antivirals as well as mutual competition for binding sites cannot be excluded. To overcome these caveats, we assessed differential scanning fluorimetry with a nanoDSF device as a label-free alternative. NanoDSF monitors the changes in the intrinsic tryptophan fluorescence (ITF) resulting from alterations of the 3D-structure of proteins as a function of the temperature. Using rhinovirus A2 as a model, we demonstrate that nanoDFS is well suited for recording the temperature-dependence of conformational changes associated with viral uncoating with minute amounts of sample. We compare it with orthogonal methods and correlate the increase in viral RNA exposure with PaSTRy measurements. Importantly, nanoDSF correctly identified the thermal stabilization of RV-A2 by pleconaril, a prototypic pocket-binding antiviral compound. NanoDFS is thus a label-free, high throughput-customizable, attractive alternative for the discovery of capsid-binding compounds impacting on viral stability.

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“…Since its first reference in the literature by Kaufman et al in 1996 [ 4 ], gas-phase electrophoresis has been applied for the characterization of a multitude of (bio-)nanoparticle materials, such as viruses [ 11 , 12 , 13 ], VLPs [ 14 , 15 , 16 , 17 ], liposomes and lipoprotein particles [ 18 , 19 , 20 , 21 , 22 ], extracellular vesicles [ 23 , 24 , 25 ], and organic and inorganic nanoparticles [ 26 , 27 , 28 ]. Frequently, ammonium acetate is employed as an electrolyte solution for the nES process.…”

Section: Introductionmentioning

confidence: 99%

Adeno-Associated Virus-like Particles’ Response to pH Changes as Revealed by nES-DMA

Zoratto

Heuser

Friedbacher

et al. 2023

Viruses

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Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte’s pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle’s shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.

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Monolithic anion-exchange chromatography yields rhinovirus of high purity

Cited by 10 publications

References 38 publications

<p>Grazoprevir/elbasvir in peginterferon alfa plus ribavirin experienced patients with chronic genotype 1 HCV/HIV co-infection: a non-randomized, open-label clinical trial</p>

<p>Grazoprevir/elbasvir in peginterferon alfa plus ribavirin experienced patients with chronic genotype 1 HCV/HIV co-infection: a non-randomized, open-label clinical trial</p>

nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

Adeno-Associated Virus-like Particles’ Response to pH Changes as Revealed by nES-DMA

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