Binding of Coxsackievirus A21 procapsids to immobilized glutathione depends on cell culture conditions during infection

Swartz, Andrew R.; Shieh, Yvonne; Gulasarian, Amanda; Olson, Jessica; Rustandi, Richard R.

doi:10.1016/j.virol.2022.06.013

Cited by 5 publications

(11 citation statements)

References 34 publications

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“…GSH, therefore, stabilises only the native D-antigenic state and D→C conversion would be expected to eject both GSH and the pocket factor. Similar conformational changes between two particle states are seen in other GSH-binding enteroviruses, and thus the use of GSH affinity columns for enterovirus purification will likely select for the D-antigenic states and could lead to loss/removal of material for less stable viruses and VLPs 37 .…”

Section: Discussionmentioning

confidence: 85%

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

et al. 2022

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Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

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Section: Discussionmentioning

confidence: 85%

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

et al. 2022

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“…In the latter, the pocket is opened by approximately 5 Å so that the key stabilising interactions cannot be made, explaining the observed stabilisation of the D-antigenic state by GSH. Similar conformational changes between two particle states are seen in other GSH-binding enteroviruses and thus the use of GSH affinity columns for enterovirus purification will likely select for the D-antigenic states and could lead to loss/removal of material for less stable viruses and VLPs 35 .…”

Section: Discussionmentioning

confidence: 84%

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

Bahar

Nasta

Fox

et al. 2022

Preprint

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“…A prototype strain of coxsackievirus A21 (CVA21), a nonenveloped enterovirus in the Picornaviridae family, is undergoing clinical evaluation as an oncolytic virus immunotherapy application for the treatment of several types of cancers (Bradley et al, 2014;Annels et al, 2019;Andtbacka et al, 2021). Similar to conventional viral vector production (Segura et al, 2011), early batches of CVA21 were purified in small quantities from static MRC-5 cell culture by gradient ultracentrifugation for clearance of upstream impurities including bovine serum albumin (BSA), host cell DNA (HC-DNA), and empty viral capsids (Swartz et al, 2022). Scalable upstream and downstream CVA21 production technologies were investigated including microcarrier stirred-tank bioreactors and affinity chromatography to replace static cell culture and ultracentrifugation (Swartz et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

“…Similar to conventional viral vector production (Segura et al, 2011), early batches of CVA21 were purified in small quantities from static MRC-5 cell culture by gradient ultracentrifugation for clearance of upstream impurities including bovine serum albumin (BSA), host cell DNA (HC-DNA), and empty viral capsids (Swartz et al, 2022). Scalable upstream and downstream CVA21 production technologies were investigated including microcarrier stirred-tank bioreactors and affinity chromatography to replace static cell culture and ultracentrifugation (Swartz et al, 2022). The growth of adherent cells on suspended microcarrier beads in stirred-tank bioreactors has been demonstrated for the production of other viruses (Wu et al, 2004;Trabelsi et al, 2012), but there was no known commercially available affinity chromatography ligand for CVA21.…”

Section: Introductionmentioning

confidence: 99%

“…Based on reports that enteroviruses interact with intracellular antioxidant glutathione (GSH) during capsid assembly in infected cells (Ma et al, 2014;Thibaut et al, 2014;Duyvesteyn et al, 2020), we developed a selective CVA21 affinity chromatography method by repurposing commercially available immobilized GSH resin originally intended for glutathione S-transferase (GST)-tagged recombinant protein purification (Swartz et al, 2022). Infectious, genome-containing capsids bound and eluted with high yield from all clarified harvests tested, but non-infectious, empty capsid binding to the GSH ligand was found to depend on the cell culture conditions during infection.…”

Section: Introductionmentioning

confidence: 99%

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Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

Swartz

Shieh

Gulasarian

et al. 2023

Front. Bioeng. Biotechnol.

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Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two–three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

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Binding of Coxsackievirus A21 procapsids to immobilized glutathione depends on cell culture conditions during infection

Cited by 5 publications

References 34 publications

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

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