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

Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides▿

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection

Synthetic Ultrashort Cationic Lipopeptides Induce Systemic Plant Defense Responses against Bacterial and Fungal Pathogens ▿

A new family of synthetic, membrane-active, ultrashort lipopeptides composed of only four amino acids linked to fatty acids was tested for the ability to induce systemic resistance and defense responses in plants. We found that two peptides wherein the third residue is a d-enantiomer (italic), C16-KKKK and C16-KLLK, can induce medium alkalinization of tobacco suspension-cultured cells and expression of defense-related genes in cucumber and Arabidopsis seedlings. Moreover, these compounds can prime systemic induction of antimicrobial compounds in cucumber leaves similarly to the plant-beneficial fungus Trichoderma asperellum T203 and provide systemic protection against the phytopathogens Botrytis cinerea B05, Pseudomonas syringae pv. lachrimans, and P. syringae pv. tomato DC3000. Thus, short cationic lipopeptides are a new category of compounds with potentially high utility in the induction of systemic resistance in plants

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

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs), which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50) of botulinum toxin, whereas the F(ab′)2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′)2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins