Enhanced production yields of rVSV-SARS-CoV-2 vaccine using Fibra-Cel® macrocarriers

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms

Role of the P–F Bond in Fluoride-Promoted Aqueous VX Hydrolysis: An Experimental and Theoretical Study

Following our ongoing studies on the reactivity of the fluoride ion toward organophosphorus compounds, we established that the extremely toxic and environmentally persistent chemical warfare agent VX (<i>O</i>-ethyl <i>S</i>-2-(diisopropylamino)­ethyl methylphosphonothioate) is exclusively and rapidly degraded to the nontoxic product EMPA (ethyl methylphosphonic acid) even in dilute aqueous solutions of fluoride. The unique role of the P–F bond formation in the reaction mechanism was explored using both experimental and computational mechanistic studies. In most cases, the “G-analogue” (<i>O</i>-ethyl methylphosphonofluoridate, Et-G) was observed as an intermediate. Noteworthy and of practical importance is the fact that the toxic side product desethyl-VX, which is formed in substantial quantities during the slow degradation of VX in unbuffered water, is completely avoided in the presence of fluoride. A computational study on a VX-model, <i>O</i>,<i>S</i>-diethyl methylphosphonothioate (<b>1</b>), clarifies the distinctive tendency of aqueous fluoride ions to react with such organophosphorus compounds. The facility of the degradation process even in dilute fluoride solutions is due to the increased reactivity of fluoride, which is caused by the significant low activation barrier for the P–F bond formation. In addition, the unique nucleophilicity of fluoride versus hydroxide toward VX, in contrast to their relative basicity, is discussed. Although the reaction outcomes were similar, much slower reaction rates were observed experimentally for the VX-model (<b>1</b>) in comparison to VX

Component Mobility by a Minute Quantity of the Appropriate Solvent as a Principal Motif in the Acceleration of Solid-Supported Reactions

The effects solvents have on fluoride-promoted heterogeneous hydrolysis and alcoholysis of various organo-phosphorus (OP) compounds on the surface of KF/Al₂O₃ are described. Solid-state magic angle spinning NMR analyses and SEM microscopy have shown that not only is the identity of the solvent important in these reactions but also its quantity. That is, minimal solvent amounts are favored and much more effective in such solid-supported reactions (and maybe generally) than those featuring solvent-free or excess solvent (>50 wt %) conditions. The addition of a minute quantity of the correct solvent (3–10 wt %, molar equivalent scale) avoids reagents leaching from the matrix, permits mobility (mass transport) of the reaction components and ensures their very high local concentration in close proximity to the solid-support large porous surface area. Accordingly, significant acceleration of reactions rates by orders of magnitude is obtained. Fascinatingly, even challenging phosphoesters with poor leaving groups, which were found to be very stable in the presence of solvent-free KF/Al₂O₃ or wetted with excess water, were efficiently hydrolyzed with a minute amount of this solvent

A novel swine model of ricin-induced acute respiratory distress syndrome

Pulmonary exposure to the plant toxin ricin leads to respiratory insufficiency and death. To date, in-depth study of acute respiratory distress syndrome (ARDS) following pulmonary exposure to toxins is hampered by the lack of an appropriate animal model. To this end, we established the pig as a large animal model for the comprehensive study of the multifarious clinical manifestations of pulmonary ricinosis. Here, we report for the first time, the monitoring of barometric whole body plethysmography for pulmonary function tests in non-anesthetized ricin-treated pigs. Up to 30 h post-exposure, as a result of progressing hypoxemia and to prevent carbon dioxide retention, animals exhibited a compensatory response of elevation in minute volume, attributed mainly to a large elevation in respiratory rate with minimal response in tidal volume. This response was followed by decompensation, manifested by a decrease in minute volume and severe hypoxemia, refractory to oxygen treatment. Radiological evaluation revealed evidence of early diffuse bilateral pulmonary infiltrates while hemodynamic parameters remained unchanged, excluding cardiac failure as an explanation for respiratory insufficiency. Ricin-intoxicated pigs suffered from increased lung permeability accompanied by cytokine storming. Histological studies revealed lung tissue insults that accumulated over time and led to diffuse alveolar damage. Charting the decline in PaO2/FiO2 ratio in a mechanically ventilated pig confirmed that ricin-induced respiratory damage complies with the accepted diagnostic criteria for ARDS. The establishment of this animal model of pulmonary ricinosis should help in the pursuit of efficient medical countermeasures specifically tailored to deal with the respiratory deficiencies stemming from ricin-induced ARDS

Capivasertib in Hormone Receptor-Positive Advanced Breast Cancer.

Background: AKT pathway activation is implicated in endocrine-therapy resistance. Data on the efficacy and safety of the AKT inhibitor capivasertib, as an addition to fulvestrant therapy, in patients with hormone receptor-positive advanced breast cancer are limited. Methods: In a phase 3, randomized, double-blind trial, we enrolled eligible pre-, peri-, and postmenopausal women and men with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer who had had a relapse or disease progression during or after treatment with an aromatase inhibitor, with or without previous cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor therapy. Patients were randomly assigned in a 1:1 ratio to receive capivasertib plus fulvestrant or placebo plus fulvestrant. The dual primary end point was investigator-assessed progression-free survival assessed both in the overall population and among patients with AKT pathway-altered (PIK3CA, AKT1, or PTEN) tumors. Safety was assessed. Results: Overall, 708 patients underwent randomization; 289 patients (40.8%) had AKT pathway alterations, and 489 (69.1%) had received a CDK4/6 inhibitor previously for advanced breast cancer. In the overall population, the median progression-free survival was 7.2 months in the capivasertib-fulvestrant group, as compared with 3.6 months in the placebo-fulvestrant group (hazard ratio for progression or death, 0.60; 95% confidence interval [CI], 0.51 to 0.71; P&lt;0.001). In the AKT pathway-altered population, the median progression-free survival was 7.3 months in the capivasertib-fulvestrant group, as compared with 3.1 months in the placebo-fulvestrant group (hazard ratio, 0.50; 95% CI, 0.38 to 0.65; P&lt;0.001). The most frequent adverse events of grade 3 or higher in patients receiving capivasertib-fulvestrant were rash (in 12.1% of patients, vs. in 0.3% of those receiving placebo-fulvestrant) and diarrhea (in 9.3% vs. 0.3%). Adverse events leading to discontinuation were reported in 13.0% of the patients receiving capivasertib and in 2.3% of those receiving placebo. Conclusions: Capivasertib-fulvestrant therapy resulted in significantly longer progression-free survival than treatment with fulvestrant alone among patients with hormone receptor-positive advanced breast cancer whose disease had progressed during or after previous aromatase inhibitor therapy with or without a CDK4/6 inhibitor. (Funded by AstraZeneca and the National Cancer Institute; CAPItello-291 ClinicalTrials.gov number, NCT04305496.)