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.

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 (<15%), while the use of packed-bed chromatography, where the surface area is smaller, achieves better recovery (up to 33%). Finally, a highly efficient chromatography purification process with CaptoTM 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.

To face the coronavirus disease 2019 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus, our institute has developed the rVSV‐ΔG‐spike vaccine, in which the glycoprotein of vesicular stomatitis virus (VSV) was replaced by the spike protein of SARS‐CoV‐2. Many process parameters can influence production yield. To maximize virus vaccine yield, each parameter should be tested independently and in combination with others. Here, we report the optimization of the production of the VSV‐ΔG‐spike vaccine in Vero cells using the Ambr15 system. This system facilitates high‐throughput screening of process parameters, as it contains 24 individually controlled, single‐use stirred‐tank minireactors. During optimization, critical parameters were tested. Those parameters included: cell densities; the multiplicity of infection; virus production temperature; medium addition and medium exchange; and supplementation of glucose in the virus production step. Virus production temperature, medium addition, and medium exchange were all found to significantly influence the yield. The optimized parameters were tested in the BioBLU 5p bioreactors production process and those that were found to contribute to the vaccine yield were integrated into the final process. The findings of this study demonstrate that an Ambr15 system is an effective tool for bioprocess optimization of vaccine production using macrocarriers and that the combination of production temperature, rate of medium addition, and medium exchange significantly improved virus yield.

Botulinum neurotoxins (BoNTs) are bacterial proteins that cause botulism, a life-threatening disease. The Endopep-MS assay permits rapid detection and serotypic differential diagnosis of BoNTs. The serotype-specific nature of this assay requires that each serum sample be aliquoted and individually tested, which in addition to the limited volume of clinical samples, especially in infants, points to the need for a multiplex assay. However, previous attempts to develop such an assay have been challenging, mainly due to inhibition of BoNT/A activity by the BoNT/E peptide substrate. BoNT/A and BoNT/E share the same native target protein as their substrate. We hypothesized that the steric interference between the BoNT/A and BoNT/E substrate peptides is responsible for the difficulty in simultaneously assaying these two toxins. To explore the basis for steric interference, we used the reported structure of BoNT/A in complex with SNAP-25 and modelled the structure of BoNT/E with SNAP-25. Following this thorough structural analysis, we designed a new peptide substrate for BoNT/A that maintained the assay sensitivity and allowed, for the first time, simultaneous detection of the three most abundant human botulinum serotypes. Adopting the multiplex assay will minimize the required sample volume and assay time for botulinum detection while maintaining the superior Endopep-MS assay performance.

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