Bioanalytics for Influenza Virus-Like Particle Characterization and Process Monitoring
Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs' unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies - nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs' thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90-115 nm for NTA and 129-141 nm for TRPS), surface charges (average of -20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.
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.