Optimization of VSV-ΔG-spike production process with the Ambr15 system for a SARS-COV-2 vaccine.

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.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

A Novel Running Wheel Mouse Model for Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Antitoxin is currently the only approved therapy for botulinum intoxications. The efficacy of antitoxin preparations is evaluated in animals. However, while in practice antitoxin is administered to patients only after symptom onset, in most animal studies, it is tested in relation to time postintoxication. This may be attributed to difficulties in quantitating early botulism symptoms in animals. In the current study, a novel system based on high-resolution monitoring of mouse activity on a running wheel was developed to allow evaluation of postsymptom antitoxin efficacy. The system enables automatic and remote monitoring of 48 mice simultaneously. Based on the nocturnal activity patterns of individual naive mice, two criteria were defined as the onset of symptoms. Postsymptom treatment with a human-normalized dose of antitoxin was fully protective in mice exposed to 4 50% lethal doses (LD50s) of botulinum neurotoxin serotype A (BoNT/A) and BoNT/B. Moreover, for the first time, a high protection rate was obtained in mice treated postsymptomatically, following a challenge with BoNT/E, the fastest-acting BoNT. The running wheel system was further modified to develop a mouse model for the evaluation of next-generation therapeutics for progressive botulism at time points where antitoxin is not effective. Exposure of mice to 0.3 LD50 of BoNT/A resulted in long-lasting paralysis and a reduction in running activity for 16 to 18 days. Antitoxin treatment was no longer effective when administered 72 h postintoxication, defining the time window to evaluate next-generation therapeutics. Altogether, the running wheel systems presented herein offer quantitative means to evaluate the efficacy of current and future antibotulinum drugs.

Novel method for quantifying cells on carriers and its demonstration during SARS-2 vaccine development.

The most effective way to prevent and control infectious disease outbreak is through vaccines. The increasing use of vaccines has elevated the need to establish new manufacturing strategies. One of the major approaches is cell-based production, which creates a need for high cell density to enable higher cell production levels. This has led to development of the technology of cell carriers, including micro and macro cell carriers. To follow the production process, quantifying the number of cells on these carriers is required, as well as the tracking of their viability and proliferation. However, owing to various carriers' unique structures, tracking the cell's is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, separating cells from the carrier, staining with crystal violet, and visually counting under a microscope. This method is tedious and counts both live and dead cells. A few other techniques were developed but were specific to the carrier type and involved specialized equipment. In this study, we describe a broadly ranging method for counting cells on carriers that was developed and employed as part of the development of severe acute respiratory syndrome coronavirus 2 vaccine. The method is based on the Alamar blue dye, a well-known, common marker for cell activity, and was found to be successful in tracking cell adsorption, cell growth, and viability on carriers. No separation of the cells from the carriers is needed, nor is any specialized equipment; the method is simple and rapid and provides comprehensive details necessary for process control of viral vaccine production in cells. This method can be easily implemented in any of a number of cell-based processes and other unique platforms for measuring the growth of encapsulated cells.

New approach for the rational selection of markers to identify botulinum toxins.

The application of mass spectrometry (MS) to detect unique peptide markers has been widely employed as a means of identifying bacterial proteins. Botulinum neurotoxins (BoNTs) are bacterial proteins that cause the life-threatening disease botulism. BoNTs are divided into several antigenically distinct serotypes and several dozen subtypes. The toxins' molecular heterogeneity makes their detection highly challenging. In this study, we describe a new LC-MS/MS-based platform for the direct identification of proteins derived from various species and subspecies in a single assay, as exemplified by BoNTs. The platform employs a rational down-selection process through several steps based on a combination of bioinformatics, tryptic digestion, and LC-MS, each leads to the final panel of markers. This approach has been demonstrated for all 8 subtypes of botulinum serotype A (BoNT/A). Ab-independent and Ab-dependent assays were developed based on the identification of 4 rationally selected markers or a combination of some of them, which enables full selectivity coverage. The Ab-independent assay, which is highly simple and rapid, has a sample-to-result turnaround time of approximately 40 min and enables the identification of 500 MsLD50/mL (5 ng/mL) BoNT/A in complex environmental matrices. The Ab-dependent assay, which is based on toxin's specific enrichment, has a turnaround time of 100 min, but enables improved sensitivity (50 MsLD50/mL, 0.5 ng/mL). Both assays were verified and validated using various environmental samples. This approach can easily be expanded to other botulinum serotypes and exhibits the potential for even further extension as a highly multiplexed assay for protein-based toxins, viruses, and organisms.

A Novel Rabbit Spirometry Model of Type E Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Botulinum neurotoxins (BoNTs), the most poisonous substances known in nature, pose significant concern to health authorities. The only approved therapeutic for botulism is antitoxin. While administered to patients only after symptom onset, antitoxin efficacy is evaluated in animals mostly in relation to time postintoxication regardless of symptoms. This is most likely due to the difficulty in measuring early symptoms of botulism in animals. In this study, a rabbit spirometry model was developed to quantify early respiratory symptoms of type E botulism that were further used as a trigger for treatment. Impaired respiration, in the form of a reduced minute volume, was detected as early as 18.1 ± 2.9 h after intramuscular exposure to 2 rabbit 50% lethal doses (LD50) of BoNT serotype E (BoNT/E), preceding any visible symptoms. All rabbits treated with antitoxin immediately following symptom onset survived. Postsymptom antitoxin efficacy was further evaluated in relation to toxin and antitoxin dosages as well as delayed antitoxin administration. Our system enabled us to demonstrate, for the first time, full antitoxin protection of animals treated with antitoxin after the onset of objective and quantitative type E botulism symptoms. This model may be utilized to evaluate the efficacy of antitoxins for additional serotypes of BoNT as well as that of next-generation anti-BoNT drugs that enter affected cells and act when antitoxin is no longer effective.

Optimization of SNAP-25-derived peptide substrate for improved detection of botulinum A in the Endopep-MS assay.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in-vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples.

Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry.

Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase-liquid chromatography-tandem mass spectrometry/multiple reaction monitoring assay.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase-mass spectrometry (Endopep-MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep-MS assay. Our strategy was based on reported BoNT/B-substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.

Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase-mass spectrometry assay.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans. Endopeptidase-mass spectrometry (Endopep-MS) is used as a specific and rapid in vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific peptide substrate, and the cleavage products are then detected by MS. To further improve the sensitivity of the assay, we report here the rational design of a new substrate peptide for the detection of botulinum neurotoxin type E (BoNT/E). Our strategy was based on previously reported structural interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one order of magnitude increased sensitivity of the assay.

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.

Application of Ambr15 system for simulation of entire SARS-CoV-2 vaccine production process involving macrocarriers.

The Ambr15 system is an automated, high-throughput bioreactor platform which comprises 24 individually controlled, single-use stirred-tank reactors. This system plays a critical role in process development by reducing reagent requirements and facilitating high-throughput screening of process parameters. However, until now, the system was used to simulate processes involving cells in suspension or growing on microcarriers and has never been tested for simulating cells growing on macrocarriers. Moreover, to our knowledge, a complete production process including cell growth and virus production has never been simulated. Here, we demonstrate, for the first time, the amenability of the automated Ambr15 cell culture reactor system to simulate the entire SARS-CoV-2 vaccine production process using macrocarriers. To simulate the production process, accessories were first developed to enable insertion of tens of Fibra-Cel macrocarries into the reactors. Vero cell adsorption to Fibra-Cels was then monitored and its adsorption curve was studied. After incorporating of all optimized factors, Vero cells were adsorbed to and grown on Fibra-Cels for several days. During the process, culture medium was exchanged, and the quantity and viability of the cells were followed, resulting in a typical growth curve. After successfully growing cells for 6 days, they were infected with the rVSV-ΔG-Spike vaccine virus. The present results indicate that the Ambr15 system is not only suitable for simulating a process using macrocarriers, but also to simulate an entire vaccine production process, from cell adsorption, cell growth, infection and vaccine virus production.

Poly(vinyl alcohol)-methacrylate with CRGD peptide: A photocurable biocompatible hydrogel.

Polyvinyl alcohol (PVA)-based hydrogels are promising biomaterials for tissue engineering printing applications. However, one of their main disadvantages is their inability to support cell attachment, which is a critical feature for the preparation of biological scaffolds. The goal of this study was to develop a printable, cell-supportive PVA-based bioink with tunable mechanical properties, without using animal-derived polymers which potentially harbor human pathogens. An ultraviolet light (UV) curable PVA-methacrylate (PVA-MA) polymer mixed with Cys-Arg-Gly-Asp (CRGD) peptide was developed. This peptide holds the integrin receptor binding sequence - RGD, that can enhance cell attachment. The additional cysteine was designed to enable its thiol binding under UV to methacrylate groups of the UV curable PVA-MA. Vero cell, as an adherent cell model was used to assess the hydrogel's cell adhesion. It was found that the PVA-MA-CRGD formula enables the preparation of hydrogels with excellent cell attachment and had even shown superior cell attachment properties relative to added gelatin. Adding hyaluronic acid (HA) as a rheologic modulator enabled the printing of this new formula. Our overall data demonstrates the applicability of this mixture as a bioink for soft tissue engineering such as skin, adipose, liver or kidney tissue.

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine.

The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3-0.5 µg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.

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.

A Simple Method for in situ Quantification of Cells on Carriers.

The technology of cell carriers was developed as a response to the need for high cell density to enable higher production levels in cell-based production processes. To follow the production process, quantifying the number of cells on these carriers is required, as well as tracking their viability and proliferation. However, owing to various carriers' unique structures, tracking the cells is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, which is tedious and counts both live and dead cells. A few other techniques have been developed, but they are all specific to a carrier type and involve specialized equipment. Here, we describe a broad ranging method for counting cells on carriers. The method is based on the Alamar blue dye, a well-known, common marker for cell activity. No separation of the cells from the carriers is needed, nor is any specialized equipment. The method is simple and rapid, and provides comprehensive details necessary for control of production processes in cells. This method can be easily implemented in any cell-based process and other unique platforms for measuring growth of cells. Graphic abstract: Schematic of the in situ quantification method.

Phosphorescent palladium-tetrabenzoporphyrin indicators for immunosensing of small molecules with a novel optical device.

Small-molecule detection is important for many applications including clinical diagnostics, drug discovery, environmental screening, and food technology. Current techniques suffer from various limitations including cost, complex sample processing, massive instrumentation, and need for expertise. To overcome these limitations, a new optical immunosensing assay for the detection of small molecules was developed and assessed with the targets estrone (E1) and estradiol (E2). For this purpose, phosphorescent indicators were designed based on the tetrabenzoporphyrin skeleton directly linked to E1 or E2, or attached through a linker, with phosphorescence lifetimes in the range of ~100-~300 µs. The assay is an indicator displacement assay (IDA). The best performances of our optical immunosensor were obtained with the indicators E1-L-Por and E2-L-Por. As they bound to specific polyclonal antibodies, their phosphorescence (τ ~200 µs) was quenched. When an endogenous competitor was added, the indicator was displaced, and the phosphorescence was immediately recovered. These effects were measured with a new optical device, described here, and able to detect picograms of luminescent molecules emitting in the NIR range, simply by measuring phosphorescence decay. This radical switch-off/switch on process demonstrates that E1-L-Por and E2-L-Por are good candidates for in vivo and in vitro immunosensing of E1 and E2. Importantly, the present immunosensing assay can be easily adapted to other small molecules such as other hormones and drugs.

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

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.

A Rabbit Model for Prolonged Continuous Intravenous Infusion Via a Peripherally Inserted Central Catheter.

Medical treatment may require the continuous intravenous (IV) infusion of drugs to sustain the therapeutic blood concentration and to minimize dosing errors. Animal disease models that ultimately mimic the intended use of new potential drugs via a continuous IV infusion in unrestrained, free roaming animals are required. While peripherally inserted central catheters (PICCs) and other central line techniques for prolonged IV infusion of drugs are prevalent in the clinic, continuous IV infusion methods in an animal model are challenging and limited. In most cases, continuous IV infusion methods require surgical knowledge as well as expensive and complicated equipment. In the current work, we established a novel rabbit model for prolonged continuous IV infusion by inserting a PICC line from the marginal ear vein to the superior vena cava and connecting it to an externally carried ambulatory infusion pump. Either saline or a clinically relevant formulation could be steadily and continuously infused at 3-6 ml/h for 11 consecutive days into freely moving rabbits while maintaining normal body temperature, weight, and respiration physiology, as determined by daily spirometry. This new model is simple to execute and can advance the ability to administer and test new drug candidates.