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

NEURAL NETWORK CONTROL OF SWITCH MODE SYSTEMS: OFF LINE TRAINING BY AN "IDEAL CONTROLLER " DATA SET

ABSTRACT-A novel method is proposed for designing Neural Network Controllers (NNC) for switch mode systems. The method applies a new concepl: "The Ideal Controller " which is run 'off-line ' to generate a record of 'perfect ' control signals in response to input and output perturbations. The record is then used as an 'off-line' training set for a Neural Network controller. The advantages of the proposed melhod are three fold: (a) the training set is the best possible, (b) training is done 'offline' by simulation and (c) there is no need to derive or guess the control law.The present study demonstrates by simulation the potential excellent performance of a NNC for DC-DC Switch-Mode converters when trained by the proposed methodology. The proposed methodology can be readily expanded to other Switch-Modc systems such a

A UNIFIED BEHAVIORAL AVERAGE MODEL OF SEPIC CONVERTERS WITH COUPLED INDUCTORS

Abstract- An average model of SEPIC converters with coupled or uncoupled inductors was developed and verified against cycle-by-cycle simulations. The model can be used as-is by any modern circuit simulator to run steady state (DC), large signal (transient) and small signal (AC) analyses. The leakage inductances were taken into account and treated separately from the mutual inductance. The primary and secondary coupling coefficients were incorporated as parameters in the model. The coupling coefficients can be set to a value from zero to almost one, representing the complete range of possible SEPIC topologies. I

Nutritional Recommendations for Cardiovascular Disease Prevention

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Osmotic pressure between arbitrarily charged planar surfaces: A revisited approach

The properties of ionic solutions between charged surfaces are often studied within the Poisson-Boltzmann framework, by finding the electrostatic potential profile. For example, the osmotic pressure between two charged planar surfaces can be evaluated by solving coupled equations for the electrostatic potential and osmotic pressure. Such a solution relies on symmetry arguments and is restricted to either equally or oppositely charged surfaces. Here, we provide a different and more efficient scheme to derive the osmotic pressure straightforwardly, without the need to find the electrostatic potential profile. We derive analytical expressions for the osmotic pressure in terms of the inter-surface separation, salt concentration, and arbitrary boundary conditions. Such results should be useful in force measurement setups, where the force is measured between two differently prepared surfaces, or between two surfaces held at a fixed potential difference. The proposed method can be systematically used for generalized Poisson-Boltzmann theories in planar geometries, as is demonstrated for the sterically modified Poisson-Boltzmann theory

Virus Inactivation in Water Using Laser-Induced Graphene Filters

Graphene in the form of laser-induced graphene (LIG) has antimicrobial and antifouling surface effects due to its electrochemical properties and texture, and LIG-based water filters were used for the inactivation of bacteria. However, the antiviral activity of LIGbased filters has not been explored. Here we showed that LIG filters also have antiviral effects under application of electrical potential using the model prototypic poxvirus virus Vaccinia lister. This antiviral activity of the LIG filters was compared with its antibacterial activity, which showed that higher voltages are required for virus inactivation compared to bacteria. The generation of reactive oxygen species, along with surface electrical effects, play a role in the mechanism for the virus inactivation

Immunologic and Protective Properties of Subunit- vs. Whole Toxoid-Derived Anti-Botulinum Equine Antitoxin

Botulism is a paralytic disease caused by botulinum neurotoxins (BoNTs). Equine antitoxin is currently the standard therapy for botulism in human. The preparation of equine antitoxin relies on the immunization of horses with botulinum toxoid, which suffers from low yield and safety limitations. The Hc fragment of BoNTs was suggested to be a potent antibotulinum subunit vaccine. The current study presents a comparative evaluation of equine-based toxoid-derived antitoxin (TDA) and subunit-derived antitoxin (SDA). The potency of recombinant Hc/A, Hc/B, and Hc/E in mice was similar to that of toxoids of the corresponding serotypes. A single boost with Hc/E administered to a toxoid E-hyperimmune horse increased the neutralizing antibody concentration (NAC) from 250 to 850 IU/mL. Immunization of naïve horses with the recombinant subunits induced a NAC comparable to that of horses immunized with the toxoid. SDA and TDA bound common epitopes on BoNTs, as demonstrated by an in vitro competition binding assay. In vivo, SDA and TDA showed similar efficacy when administered to guinea pigs postexposure to a lethal dose of botulinum toxins. Collectively, the results of the current study suggest that recombinant BoNT subunits may replace botulinum toxoids as efficient and safe antigens for the preparation of pharmaceutical anti-botulinum equine antitoxins

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