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

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors

AF710B, a novel M1/σ1 agonist with therapeutic efficacy in animal models of Alzheimer's disease

We previously developed orthosteric M1 muscarinic agonists (e.g. AF102B, AF267B and AF292), which act as cognitive enhancers and potential disease modifiers. We now report on a novel compound, AF710B, a highly potent and selective allosteric M1 muscarinic and σ1 receptor agonist. AF710B exhibits an allosteric agonistic profile on the M1 muscarinic receptor; very low concentrations of AF710B significantly potentiated the binding and efficacy of carbachol on M1 receptors and their downstream effects (p-ERK1/2, p-CREB). AF710B (1-30 µg/kg, p.o.) was a potent and safe cognitive enhancer in rats treated with the M1 antagonist trihexyphenidyl (passive avoidance impairment). These effects of AF710B involve σ1 receptor activation. In agreement with its antiamnesic properties, AF710B (at 30 nM), via activation of M1 and a possible involvement of σ1 receptors, rescued mushroom synapse loss in PS1-KI and APP-KI neuronal cultures, while AF267B (1 µM) was less potent in PS1-KI and ineffective in APP-KI models, respectively. In female 3xTg-AD mice, AF710B (10 µg/kg, i.p./daily/2 months) (i) mitigated cognitive impairments in the Morris water maze; (ii) decreased BACE1, GSK3β activity, p25/CDK5, neuroinflammation, soluble and insoluble Aβ40, Aβ42, plaques and tau pathologies. AF710B differs from conventional σ1 and M1 muscarinic (orthosteric, allosteric or bitopic) agonists. These results highlight AF710B as a potential treatment for Alzheimer's disease (e.g. improving cognitive deficits, synaptic loss, amyloid and tau pathologies, and neuroinflammation) with a superior profile over a plethora of other therapeutic strategies

Highly Efficient Purification of Recombinant VSV-∆G-Spike Vaccine against SARS-CoV-2 by Flow-Through Chromatography

This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Several purification strategies are evaluated using a variety of chromatography methods, including membrane adsorbers and packed-bed ion-exchange chromatography. Cell harvest is initially treated with endonuclease, clarified, and further concentrated by ultrafiltration before chromatography purification. The use of anion-exchange chromatography in all forms results in strong binding of the virus to the media, necessitating a high salt concentration for elution. The large virus and spike protein binds very strongly to the high surface area of the membrane adsorbents, resulting in poor virus recovery (TM Core 700 resin, which does not require binding and the elution of the virus, is described. rVSV-S cannot enter the inner pores of the resin and is collected in the flow-through eluent. Purification of the rVSV-S virus with CaptoTM Core 700 resulted in viral infectivity above 85% for this step, with the efficient removal of host cell proteins, consistent with regulatory requirements. Similar results were obtained without an initial ultrafiltration step