RESUMEN

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.

RESUMEN

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.

Asunto(s)

Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Descubrimiento de Drogas , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Animales , Antivirales/uso terapéutico , Ácido Aurintricarboxílico/farmacología , Ácido Aurintricarboxílico/uso terapéutico , COVID-19/virología , Chlorocebus aethiops , Ácido Elágico/farmacología , Ácido Elágico/uso terapéutico , Heparina/farmacología , Heparina/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Dominios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Internalización del Virus/efectos de los fármacos

RESUMEN

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-{Delta}G-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-{Delta}G-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-{Delta}G-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-{Delta}G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

RESUMEN

The need for antiviral drugs is real and relevant. Broad spectrum antiviral drugs have a particular advantage when dealing with rapid disease outbreaks, such as the current COVID-19 pandemic. Since viruses are completely dependent on internal cell mechanisms, they must cross cell membranes during their lifecycle, creating a dependence on processes involving membrane dynamics. Thus, in this study we examined whether the synthesis of glycosphingolipids, biologically active components of cell membranes, can serve as an antiviral therapeutic target. We examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS); (i) Genz-123346, an analogue of the FDA-approved drug Cerdelga(R), (ii) GENZ-667161, an analogue of venglustat which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route: (i) Neuroinvasive Sindbis virus (SVNI), (ii) West Nile virus (WNV), (iii) Influenza A virus, and (iv) SARS-CoV-2. Moreover, GCS inhibitors significantly increase the survival rate of SVNI-infected mice. Our data suggest that GCS inhibitors can potentially serve as a broad-spectrum antiviral therapy and should be further examined in preclinical and clinical trial. Analogues of the specific compounds tested have already been studied clinically, implying they can be fast-tracked for public use. With the current COVID-19 pandemic, this may be particularly relevant to SARS-CoV-2 infection. One Sentence SummaryAn analogue of Cerdelga(R), an FDA-approved drug, is effective against a broad range of RNA-viruses including the newly emerging SARS-CoV-2.