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.

Phage Therapy Potentiates Second-Line Antibiotic Treatment against Pneumonic Plague.

Plague pandemics and outbreaks have killed millions of people during the history of humankind. The disease, caused by the bacteria Yersinia pestis, is currently treated effectively with antibiotics. However, in the case of multidrug-resistant (MDR) bacteria, alternative treatments are required. Bacteriophage (phage) therapy has shown efficient antibacterial activity in various experimental animal models and in human patients infected with different MDR pathogens. Here, we evaluated the efficiency of фA1122 and PST phage therapy, alone or in combination with second-line antibiotics, using a well-established mouse model of pneumonic plague. Phage treatment significantly delayed mortality and limited bacterial proliferation in the lungs. However, the treatment did not prevent bacteremia, suggesting that phage efficiency may decrease in the circulation. Indeed, in vitro phage proliferation assays indicated that blood exerts inhibitory effects on lytic activity, which may be the major cause of treatment inefficiency. Combining phage therapy and second-line ceftriaxone treatment, which are individually insufficient, provided protection that led to the survival of all infected animals-a synergistic protective effect that represents a proof of concept for efficient combinatorial therapy in an emergency event of a plague outbreak involving MDR Y. pestis strains.

Evaluation of a downstream process for the recovery and concentration of a Cell-Culture-Derived rVSV-Spike COVID-19 vaccine candidate.

rVSV-Spike (rVSV-S) is a recombinant viral vaccine candidate under development to control the COVID-19 pandemic and is currently in phase II clinical trials. rVSV-S induces neutralizing antibodies and protects against SARS-CoV-2 infection in animal models. Bringing rVSV-S to clinical trials required the development of a scalable downstream process for the production of rVSV-S that can meet regulatory guidelines. The objective of this study was the development of the first downstream unit operations for cell-culture-derived rVSV-S, namely, the removal of nucleic acid contamination, the clarification and concentration of viral harvested supernatant, and buffer exchange. Retaining the infectivity of the rVSV-S during the downstream process was challenged by the shear sensitivity of the enveloped rVSV-S and its membrane protruding spike protein. Through a series of screening experiments, we evaluated and established the required endonuclease treatment conditions, filter train composition, and hollow fiber-tangential flow filtration parameters to remove large particles, reduce the load of impurities, and concentrate and exchange the buffer while retaining rVSV-S infectivity. The combined effect of the first unit operations on viral recovery and the removal of critical impurities was examined during scale-up experiments. Overall, approximately 40% of viral recovery was obtained and the regulatory requirements of less than 10 ng host cell DNA per dose were met. However, while 86-97% of the host cell proteins were removed, the regulatory acceptable HCP levels were not achieved, requiring subsequent purification and polishing steps. The results we obtained during the scale-up experiments were similar to those obtained during the screening experiments, indicating the scalability of the process. The findings of this study set the foundation for the development of a complete downstream manufacturing process, requiring subsequent purification and polishing unit operations for clinical preparations of rVSV-S.

Virus Inactivation in Water Using Laser-Induced Graphene Filters.

Interest in the pathogenesis, detection, and prevention of viral infections has increased broadly in many fields of research over the past year. The development of water treatment technology to combat viral infection by inactivation or disinfection might play a key role in infection prevention in places where drinking water sources are biologically contaminated. Laser-induced graphene (LIG) has antimicrobial and antifouling surface effects mainly because of its electrochemical properties and texture, and LIG-based water filters have been used for the inactivation of bacteria. However, the antiviral activity of LIG-based filters has not yet been explored. Here we show that LIG filters also have antiviral effects by applying electrical potential during filtration of the model prototypic poxvirus Vaccinia lister. This antiviral activity of the LIG filters was compared with its antibacterial activity, which showed that higher voltages were required for the inactivation of viruses compared to that of bacteria. The generation of reactive oxygen species, along with surface electrical effects, played a role in the mechanism of virus inactivation. This new property of LIG highlights its potential for use in water and wastewater treatment for the electrochemical disinfection of various pathogenic microorganisms, including bacteria and viruses.

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 (<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.

In-Line Monitoring of Downstream Purification Processes for VSV Based SARS-CoV-2 Vaccine Using a Novel Technique.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.

Bulging fontanelle in febrile infants as a predictor of bacterial meningitis.

It is common practice to perform a lumbar puncture in infants presenting with fever and a bulging fontanelle in order to rule out bacterial meningitis. However, most of these infants have benign, self-limiting diseases. The objective was to determine whether there is an association between bulging fontanelle and bacterial meningitis in febrile infants. This retrospective cohort study included febrile children with a bulging fontanelle who underwent lumbar puncture at Meir Medical Center from 2005 through 2015. A total of 764 children ages 2-18 months underwent lumbar puncture during the study period. Among them, 304 had a bulging fontanelle and fever on evaluation and cerebrospinal fluid pleocytosis was found in 115 (37.8%), including 1 case of bacterial meningitis (0.3%). None of the infants described on admission as appearing well on presentation was found to have bacterial meningitis. Of the 764 children who underwent lumbar puncture, 10 infants were diagnosed with bacterial meningitis, and only one (10%) presented with a bulging fontanelle.Conclusion: The finding of a bulging fontanelle has very low sensitivity and specificity for bacterial meningitis. Most causes of a bulging fontanelle in febrile infants are self-limiting diseases. The routine approach of performing a lumbar puncture in febrile infants with a bulging fontanelle should be reconsidered. What is Known: • It is common to perform a lumbar puncture in febrile infants with a bulging fontanelle, to rule out bacterial meningitis. • However, there are only few researches regarding the relationship between bulging fontanelle and bacterial meningitis. What is New: • The finding of a bulging fontanelle has very low sensitivity and specificity for bacterial meningitis • The need for routine lumbar puncture in these cases should be reconsidered.

Sensitive Readout for Microfluidic High-Throughput Applications using Scanning SQUID Microscopy.

Microfluidic chips provide a powerful platform for high-throughput screening of diverse biophysical systems. The most prevalent detection methods are fluorescence based. Developing new readout techniques for microfluidics focusing on quantitative information in the low signal regime is desirable. In this work, we combine the well-established immunoassay approach, with magnetic nanoparticles, with a highly sensitive magnetic imaging technique. We offer to integrate a microfluidic array into a scanning superconducting quantum interference device (SQUID) microscope, to image nanoparticles that were moved through the microfluidic device. We demonstrate the technique on protein-protein interactions (PPI). We compare sensitivity to that of a conventional readout, quantify the amount of interactions, and demonstrate 0.1 atto-mole sensitivity. Our work serves as a proof of concept that will promote the development of a new set of eyes, a stable usable microfluidic-scanning SQUID microscopy.

Characterization of peptides self-assembly by low frequency Raman spectroscopy.

Low Frequency Vibrational (LFV) modes of peptides and proteins are attributed to the lattice vibrations and are dependent on their structural organization and self-assembly. Studies taken in order to assign specific absorption bands in the low frequency range to self-assembly behavior of peptides and proteins have been challenging. Here we used a single stage Low Frequency Raman (LF-Raman) spectrometer to study a series of diastereomeric analogue peptides to investigate the effect of peptides self-assembly on the LF-Raman modes. The structural variation of the diastereomeric analogues resulted in distinct self-assembly groups, as confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) data. Using LF-Raman spectroscopy, we consistently observed discrete peaks for each of the self-assembly groups. The correlation between the spectral features and structural morphologies was further supported by principal component analysis (PCA). The LFV modes provide further information on the degrees of freedom of the entire peptide within the higher order organization, reflecting the different arrangement of its hydrogen bonding and hydrophobic interactions. Thus, our approach provides a simple and robust complementary method to structural characterization of peptides assemblies.

New Method to Study the Vibrational Modes of Biomolecules in the Terahertz Range Based on a Single-Stage Raman Spectrometer.

The low-frequency vibrational (LFV) modes of biomolecules reflect specific intramolecular and intermolecular thermally induced fluctuations that are driven by external perturbations, such as ligand binding, protein interaction, electron transfer, and enzymatic activity. Large efforts have been invested over the years to develop methods to access the LFV modes due to their importance in the studies of the mechanisms and biological functions of biomolecules. Here, we present a method to measure the LFV modes of biomolecules based on Raman spectroscopy that combines volume holographic filters with a single-stage spectrometer, to obtain high signal-to-noise-ratio spectra in short acquisition times. We show that this method enables LFV mode characterization of biomolecules even in a hydrated environment. The measured spectra exhibit distinct features originating from intra- and/or intermolecular collective motion and lattice modes. The observed modes are highly sensitive to the overall structure, size, long-range order, and configuration of the molecules, as well as to their environment. Thus, the LFV Raman spectrum acts as a fingerprint of the molecular structure and conformational state of a biomolecule. The comprehensive method we present here is widely applicable, thus enabling high-throughput study of LFV modes of biomolecules.

An off-the-shelf integrated microfluidic device comprising self-assembled monolayers for protein array experiments.

Microfluidic-based protein arrays are promising tools for life sciences, with increased sensitivity and specificity. One of the drawbacks of this technology is the need to create fresh surface chemistry for protein immobilization at the beginning of each experiment. In this work, we attempted to include the process of surface functionalization as part of the fabrication of the device, which would substitute the time consuming step of surface functionalization at the beginning of each protein array experiment. To this end, we employed a novel surface modification using self-assembled monolayers (SAMs) to immobilize biomolecules within the channels of a polydimethylsiloxane (PDMS) integrated microfluidic device. As a model, we present a general method for depositing siloxane-anchored SAMs, with 1-undecyl-thioacetate-trichlorosilane (C11TA) on the silica surfaces. The process involved developing PDMS-compatible conditions for both SAM deposition and functional group activation. We successfully demonstrated the ability to produce, within an integrated microfluidic channel, a C11TA monolayer with a covalently conjugated antibody. The antibody could then bind its antigen with a high signal to background ratio. We further demonstrated that the antibody was still active after storage of the device for a week. Integration of the surface chemistry into the device as part of its fabrication process has potential to significantly simplify and shorten many experimental procedures involving microfluidic-based protein arrays. In turn, this will allow for broader dissemination of this important technology.

Sedation methods for intra-articular corticosteroid injections in Juvenile Idiopathic Arthritis: a review.

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children. Intra-articular corticosteroid injection (IASI), one of the cornerstones of treatment for this disease, is usually associated with anxiety and pain. IASI in JIA may be performed under general anesthesia, conscious sedation, or local anesthesia alone. Currently, there is no widely accepted standard of care regarding the sedation method for IASI. This review discusses the different methods of anesthesia and sedation in this procedure, emphasizing the advantages and shortcomings of each method.

[Medical clowns--dream doctors as important team members in the treatment of young children with juvenile idiopathic arthritis].

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children. An intra-articular corticosteroid injection (IAS), one of the cornerstones of treatment for this disease, is usuaLLy associated with anxiety and pain. A major part of the success in reducing the pain is associated with the level of the child's anxiety even before starting the procedure. This is a case study of a 5 year old girl with JIA, who has been treated with an intra-articular corticosteroid injection to her knee joint. The case study is presented from the point of view of the medical clown, who is an important integral part of the team of the IAS procedure. In this article we will discuss the participation of medical clowns in the treatment of children in general, and in the IAS procedure in particular. The importance of the subject stems from the fact that it has been proven that the presence of medical clowns significantly alleviates the children's anxiety and pain. This study, as well as others on this subject, shows that we should encourage medical clowns as an integraL part of the treatment of children.

Bone strength in children with growing pains: long-term follow-up.

OBJECTIVES: To examine the changes in bone strength in a cohort of children with 'growing pains' (GP) after 5 years follow-up and the correlation with pain outcome. METHODS: Bone strength was measured by quantitative ultrasound. Subjects were 39 children with GP previously studied. Controls were normograms based on the measurement of bone speed of sound in 1085 healthy children. Current GP status was assessed by parental questionnaires. Bone strength was compared with pain outcome. RESULTS: We examined 30/39 (77%) patients after 5 years. Bone strength was significantly increased when compared to the first study (Z score 0.65±1.77 vs. -0.62±0.90, p<0.001). While overall there was no significant difference in the bone strength between the 16 (53%) patients whose GP resolved and the 14 (47%) who continued to have GP episodes (p=0.71), all 6 (20%) patients with a speed of sound Z-score <-1 continued to have GP (p=0.003). CONCLUSIONS: Our findings that pain improves in most patients parallel to the increase in bone strength may support the hypothesis of GP representing in some patients a local overuse syndrome.

A screen identifying genes responsive to Dpp and Wg signaling in the Drosophila developing wing.

Morphogens control developmental decisions by regulating the expression of different sets of target genes in specific regions. The products of these genes drive the region specific developmental program and lead to the formation of different adult structures. Therefore, identification of the morphogens downstream target effectors is an essential step in deciphering the mechanisms underlying organ development. In the Drosophila developing wing, signaling by the morphogens Decapentaplegic (Dpp) and Wingless (Wg) plays global roles in controlling differentiation, growth, and survival. However, only a handful of their downstream target genes have been identified. Here, we describe a screening method designed to quickly search through large collections of P-Gal4 enhancer-traps for candidate genes that are transcriptionally regulated by Dpp or Wg signaling. Using this screening method, we screened a small collection of lethal P-Gal4 insertion lines that are expressed in the wing disc. We identified 10 novel putative Dpp and/or Wg target genes in the developing wing. Further study of the roles of the genes recognized in this screen should shed light on how morphogens control various processes in organ development.

Preassembly of membrane-active peptides is an important factor in their selectivity toward target cells.

Membrane-active peptides comprise a large group of toxins used in the defense and offense systems of all organisms including plants and humans. They act on diverse targets including microorganisms and mammalian cells, but the factors that determine their target cell selectivity are not yet clear. Here, we tested the role of peptide length and preassembly on the ability of diastereomeric cationic antimicrobial peptides to discriminate among bacteria, erythrocytes, and fungal cells, by using peptides with variable lengths (13, 16, and 19 amino acids long) and their covalently linked pentameric bundles. All the bundles expressed similar potent antifungal activity (minimal inhibitory concentration of 0.2-0.3 microM) and high antimicrobial activity. Hemolytic activity was also observed at concentrations higher than those required for antifungal activity. In contrast, all the monomers showed length-dependent antimicrobial activity, were less active toward bacteria and fungi, and were devoid of hemolytic activity. BIAcore biosensor experiments revealed a approximately 300-fold increase in peptide-membrane binding affinity between the 13- and 19-residue monomers toward zwitterionic (egg phosphatidylcholine (PC)/egg spingomyelin (SM)/cholesterol) vesicles. All the monomeric peptides display a similar high affinity to negatively charged (E. coli phosphatidylethanolamine (PE)/egg phosphatidylglycerol (PG)) vesicles regardless of their length. In contrast, irrespective of the size of the monomeric subunit, all the bundles bind irreversibly and strongly disrupt both PC/SM/cholesterol and PE/PG membranes. Attenuated total reflectance Fourier-transform infrared spectroscopy revealed that peptide assembly also affects structure as observed by an increased alpha-helical and beta-sheet content in membranes and enhances acyl chain disruption of PC/cholesterol. The correlation between the antibacterial activity and ability to depolarize the transmembrane potential of E. coli spheroplasts, as well as the ability to induce calcein release from vesicles, suggests that the bacterial membrane is their target. The data demonstrate that preassembly of cationic diastereomeric antimicrobial peptides is an essential factor in their membrane targeting.

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.

The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial peptide and its diastereomers.

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with l-amino acid peptides possessing amphipathic alpha-helix or beta-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic alpha-helical peptide (15 amino acids long) and its diastereomer (composed of both l- and d-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the l-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all l-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all l-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.