Oncolytic virus purification with periodic counter‐current chromatography
Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.
SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.
Marine phages have been applied to trace ground- and surface water flows. Yet, information on their transport in soil and related particle intactness is limited. Here we compared the breakthrough of two lytic marine tracer phages (Pseudoalteromonas phages PSA-HM1 and PSA-HS2) with the commonly used Escherichia virus T4 in soil- and sand-filled laboratory percolation columns. All three phages showed high mass recoveries in the effluents and a higher transport velocity than non-reactive tracer Br−. Comparison of effluent gene copy numbers (CN) to physically-determined phage particle counts or infectious phage counts showed that PSA-HM1 and PSA-HS2 retained high phage particle intactness (Ip > 81%), in contrast to T4 (Ip < 36%). Our data suggest that marine phages may be applied in soil to mimic the transport of (bio-) colloids or anthropogenic nanoparticles of similar traits. Quantitative PCR (qPCR) thereby allows for highly sensitive quantification and thus for the detection of even highly diluted marine tracer phages in environmental samples.
As of 10 December 2021, coronavirus disease 2019 (COVID‐19) caused by SARS‐CoV‐2 accounted for 267 million people with up to 5.3 million deaths worldwide (https://covid19.who.int). Since late 2019, much progress has been made in response to the COVID‐19 pandemic, including the rapid developments of effective vaccines and the treatment guidelines consisting of antiviral drugs, immunomodulators, and critical care support (https://covid19.who.int). However, SARS‐CoV‐2 evolves over time as its genome has a high mutation rate that leads to reasonable concerns of breakthrough infection due to immune escape and resistant strain emergence under antiviral pressure (Lipsitch et al., 2021; Szemiel et al., 2021). A newly emerging Omicron (B.1.1.529) variant rings alarms around the globe that, perhaps, the COVID‐19 war has just begun. Relentless efforts should be made to advance our knowledge and treatment regimens against COVID‐19. These included studies of mesenchymal stem cell (MSC) therapy that aimed to mitigate cytokine storm and promote tissue repair in severely ill patients with COVID‐19 pneumonia and acute respiratory distress syndrome (ARDS) (Hashemian et al., 2021; Meng et al., 2020; Zhu et al., 2021). Nevertheless, as extensively discussed in a recent review by Dr. Phillip W. Askenase of Yale University School of Medicine, the immunomodulatory and regenerative effects of MSC therapy are mediated through MSC‐derived extracellular vesicles (MSC‐EVs) (Askenase, 2020), while the use of MSC‐EVs has less safety concerns of thromboembolism, arrhythmia and malignant transformation. In this direction, MSC‐EV investigations for COVID‐19 treatment would be more appealing and undeniable if MSC‐EVs also exhibit anti‐SARS‐CoV‐2 effects. A previous study revealed that MSC‐EVs pertained antiviral activity against influenza virus in a preclinical model (Khatri et al., 2018). It is known that MSCs are highly resistant to viral infections (Wu et al., 2018), including SARS‐CoV‐2 (Avanzini et al., 2021). We, therefore, hypothesized that the EVs released from MSCs could inhibit SARS‐CoV‐2 infection.