SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradle(TM) 500-AP bioreactor. CelCradle(TM) 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10(8) Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2–2.5 × 10(9) cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log(10) 50% tissue culture infectious dose (TCID(50))/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log(10) TCID(50)/mL, and a total of 10.5 log(10) TCID(50) were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration–response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7–12 µg/mL), followed by artemether (53–98 µg/mL), A. annua extracts (83–260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment

Physiology of Escherichia coli at high osmolarity and its use in industrial ethanol production

Biofuels are becoming increasingly important in the light of climate change, increasing energy demands and higher fuel prices. Their production must be carefully balanced against the production of foods and use of fresh water, both of which are consumed by crop based biofuels such as corn ethanol. One proposed solution is to instead use waste materials such as plant matter including wood offcuts and plant trimmings. This waste can be turned into syngas (a mix of CO and H₂) and converted to ethanol using microorganisms. Production of ethanol using microorganisms however, is complicated as the ethanol produced by the cells becomes toxic at higher concentrations, inhibiting their growth and further production. The usual method of keeping the toxicity down to allow further production is to continuously distil ethanol off at low concentrations and consequently, a high cost. Since the mechanisms of ethanol damage to microbes are similar to those that occur during osmotic challenge: damage to the membrane, cytoplasmic dehydration, and protein unfolding, I hypothesized that we can use knowledge of osmoregulatory mechanisms to increase the resistance of cells to ethanol damage and decrease distillation costs. While working under this hypothesis I had to address some of the challenges one faces when understanding the physiology and growth of microbes, and for the purpose I have developed a number of useful techniques; a method for calibrating optical densities to cell number, a neural network for identifying cells and determining their concentrations via microscope imaging and a simple particle diffusion simulation for correcting errors due to confinement of particles within cells. In addition, I have produced a simplified model of industrial production to help evaluate economic impacts that changes to the growth of microbes and the plant process may have. To study any useful links between osmolarity and ethanol resistance, I chose to use Escherichia coli as the model organism due to the large amount of data available on its osmoregulatory mechanisms. It has been long known that when bacteria do grow at high but not lethal osmolarity, they grow at a reduced rate which, even if it increases the ethanol resistance, may have a detrimental effect on the desired production rates. So therefore, in addition to testing the ethanol tolerance of the bacteria under different osmotic conditions, and as a second focus of this project, I have tried to understand why the reduction in growth rates occurs, with the hope of mitigating this effect. This will offer a better understanding of osmotic growth and provide useful insights for industrial bio-production. To this end, I have tried to discern some of the possible reasons for this slower growth by measuring various cell physiological parameters such as batch-culture yield, cytoplasmic diffusion and proteome allocation using my newly developed techniques. I have found a reduction in the cell yield with increasing osmolarity of 50% with an increase of 1Osm of osmotic agent, a slight decrease in cytoplasmic diffusion and a slight decrease in RNA content at high osmolarity. I have also proposed a coarse-grained model of proteome partitioning to help integrate these results and explain growth at high osmolarity. It is still to be determined if, as a whole, the changes observed explain fully the reduction in growth. When it comes to ethanol resistance, and contrary to my hypothesis, I found that increasing the osmolarity of the medium with sucrose or NaCl reduced the ethanol resistance. However, I found that the proW gene provides significant ethanol resistance, indicating glycine betaine, or another substrate for this transporter, is highly useful as a protectant. And this transporter is a potential candidate for overexpression. A reduction in growth temperature also provides significant solvent tolerance at the expense of a reduction in growth rate and hence production.Restricted Acces

Efficacy of ion-channel inhibitors amantadine, memantine and rimantadine for the treatment of SARS-CoV-2 in vitro

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2