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The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than seven million deaths worldwide. To reduce viral spread, the Israel Institute for Biological Research (IIBR) developed and produced a new rVSV-SARS-CoV-2-S vaccine candidate (BriLife ® ) based on a platform of a genetically engineered vesicular stomatitis virus (VSV) vector that expresses the spike protein of SARS-CoV-2 instead of the VSV-G protein on the virus surface. Quantifying the virus titer to evaluate vaccine potency requires a reliable validated assay that meets all the stringent pharmacopeial requirements of a bioanalytical method. Here, for the first time, we present the development and extensive validation of a quantitative plaque assay using Vero E6 cells for the determination of the concentration of the rVSV-SARS-CoV-2-S viral vector. Three different vaccine preparations with varying titers (DP_low, DP_high, and QC sample) were tested according to a strict validation protocol. The newly developed plaque assay was found to be highly specific, accurate, precise, and robust. The mean deviations from the predetermined titers for the DP_low, DP_high, and QC preparations were 0.01, 0.02, and 0.09 log 10 , respectively. Moreover, the mean %CV values for intra-assay precision were 18.7%, 12.0%, and 6.0%, respectively. The virus titers did not deviate from the established values between cell passages 5 and 19, and no correlation was found between titer and passage. The validation results presented herein indicate that the newly developed plaque assay can be used to determine the concentration of the BriLife ® vaccine, suggesting that the current protocol is a reliable methodology for validating plaque assays for other viral vaccines.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cohen, Simon, Hazan, Tal, Tzadok, Levin, Girshengorn, Mimran, Natan, Baruhi, David, Rosen, Shmaya, Borni, Cohen, Lupu, Kedmi, Zilberman, Jayson, Monash, Dor, Diamant, Goldvaser, Cohen-Gihon, Israeli, Lazar, Shifman, Beth-Din, Zvi, Oren, Makovitzki, Lerer, Mimran, Toister, Zichel, Adar and Epstein.)

Background: Though rates of tobacco smoking have decreased consistently over the past 3 decades, cigarette use remains the top preventable cause of premature death in North America. The Clinical Effort Against Secondhand Smoke Exposure (CEASE) is a medical clinic-based intervention that systematically screens parents for tobacco use and offers them direct access to evidence-based smoking cessation services. While the effectiveness of CEASE for parents who smoke has already been demonstrated in the United States, the CEASE model has not yet been tested in Canada, among parents who use e-cigarettes, or among adolescents who use cigarettes and e-cigarettes., Objective: We aim to demonstrate the feasibility and evaluate the preliminary effectiveness of the CEASE program for parental smoking cessation and its adapted version for adolescent smoking cessation and adolescent and parental vaping cessation., Methods: We will approach parents or guardians of children aged between 0 and 17 years, as well as adolescent patients aged between 14 and 17 years, from a tertiary care pediatric hospital in Montreal, Quebec, Canada, for participation in this single-blinded, pilot randomized controlled trial. Eligible participants are those who report using tobacco cigarettes or e-cigarettes at least once in the last 7 days and present to an outpatient pediatric clinic for a scheduled appointment. Our recruitment target is 100 participants: 50 parents or guardians of children aged 17 years or younger, and 50 adolescents aged between 14 and 17 years. The feasibility of implementation of the CEASE model will be measured by recruitment and retention rates for all 4 participant groups (stratified as follows: parents who use cigarettes, parents who use e-cigarettes exclusively, adolescents who use cigarettes, and adolescents who use e-cigarettes exclusively). Parent and adolescent participants within each group are randomized to the intervention and control groups using a 1:1 ratio through a computer-generated randomization list. Preliminary effectiveness outcomes include self-reported smoking and e-cigarette cessation, use of cessation resources, changes in smoking and e-cigarette use, motivation to quit, and quit attempts among participants. Participants complete electronic questionnaires on a tablet in the clinic at baseline as well as electronic follow-up questionnaires at 1, 3, and 6 months. Individuals reporting successful quit attempts are invited to provide a urine sample for cotinine testing to biochemically confirm quit. Analyses include descriptive statistics as well as exploratory trajectory analyses of smoking, e-cigarette use, and motivation to quit., Results: Research activities began in June 2022. Participant enrollment and data collection began in February 2023 and are expected to be completed in 15 months., Conclusions: There is a strong need for effective and cost-effective smoking and vaping cessation interventions for parents and adolescents. If successful, this study will help inform the preparation of a fully powered randomized controlled trial of CEASE in Canada in these populations., Trial Registration: Clinicaltrials.gov NCT05366790; https://www.clinicaltrials.gov/study/NCT05366790., International Registered Report Identifier (irrid): DERR1-10.2196/47978., (©Nicholas Chadi, Emile Diamant, Tamara Perez, Afnan Al-Saleh, Marie-Pierre Sylvestre, Jennifer O’Loughlin, Jonathan P Winickoff, Olivier Drouin. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 30.11.2023.)

A novel COVID-19 vaccine (BriLife®) has been developed by the Israel Institute for Biological Research (IIBR) to prevent the spread of the SARS-CoV-2 virus throughout the population in Israel. One of the components in the vaccine formulation is tris(hydroxymethyl)aminomethane (tromethamine, TRIS), a buffering agent. TRIS is a commonly used excipient in various approved parenteral medicinal products, including the mRNA COVID-19 vaccines produced by Pfizer/BioNtech and Moderna. TRIS is a hydrophilic basic compound that does not contain any chromophores/fluorophores and hence cannot be retained and detected by reverse-phase liquid chromatography (RPLC)-ultraviolet (UV)/fluorescence methods. Among the few extant methods for TRIS determination, all exhibit a lack of selectivity and/or sensitivity and require laborious sample treatment. In this study, LC−mass spectrometry (MS) with its inherent selectivity and sensitivity in the multiple reaction monitoring (MRM) mode was utilized, for the first time, as an alternative method for TRIS quantitation. Extensive validation of the developed method demonstrated suitable specificity, linearity, precision, accuracy and robustness over the investigated concentration range (1.2−4.8 mg/mL). Specifically, the R2 of the standard curve was >0.999, the recovery was >92%, and the coefficient of variance (%CV) was <12% and <6% for repeatability and intermediate precision, respectively. Moreover, the method was validated in accordance with strict Good Manufacturing Practice (GMP) guidelines. The developed method provides valuable tools that pharmaceutical companies can use for TRIS quantitation in vaccines and other pharmaceutical products.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 (( R )-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a - c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a - c showed higher lipophilicity (higher log D 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a - c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a - c . The isopropyl ester 12c showed a promising log D 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

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.

Purpose: The purpose of this study is to compare perioperative and oncological outcomes of upfront vs. delayed early radical cystectomy (eRC) for high-risk non-muscle-invasive bladder cancer (HR-NMIBC). Methods: All consecutive HR-NMIBC patients who underwent eRC between 2001 and 2020 were retrospectively included and divided into upfront and delayed groups, according to the receipt or not of BCG. Perioperative outcomes were evaluated and the impact of upfront vs. delayed eRC on pathological upstaging, defined as ≥pT2N0 disease at final pathology, was assessed using multivariable logistic regression. Recurrence-free (RFS), cancer-specific (CSS) and overall survival (OS) were compared between upfront and delayed eRC groups using inverse probability of treatment weighting (IPTW)-adjusted Cox model. Results: Overall, 184 patients received either upfront (n = 87; 47%) or delayed (n = 97; 53%) eRC. No difference was observed in perioperative outcomes between the two treatment groups (all p > 0.05). Pathological upstaging occurred in 55 (30%) patients and upfront eRC was an independent predictor (HR = 2.65; 95% CI = (1.23−5.67); p = 0.012). In the IPTW-adjusted Cox analysis, there was no significant difference between upfront and delayed eRC in terms of RFS (HR = 1.31; 95% CI = (0.72−2.39); p = 0.38), CSS (HR = 1.09; 95% CI = (0.51−2.34); p = 0.82) and OS (HR = 1.19; 95% CI = (0.62−2.78); p = 0.60). Conclusion: our results suggest similar perioperative outcomes between upfront and delayed eRC, with an increased risk of upstaging after upfront eRC that did impact survival, as compared to delayed eRC.

The receptor-binding domain of botulinum neurotoxin (H C fragment), is a promising botulism vaccine candidate. In the current study, fermentation strategies were evaluated to upscale H C fragment expression. A simple translation of the growth conditions from shake flasks to a batch fermentation process resulted in limited culture growth and protein expression (OD of 11 and volumetric protein yields of 123 mg/L). Conducting fed-batch fermentation with rich media and continuous nutrient supplementation significantly improved culture growth (OD of 40.3) and protein expression (1093 mg/L). A further increase in H C fragment yield was achieved by high cell density cultivation (HCDC). The bacterium was grown in a defined medium and with a combined bolus/continuous feed of nutrients to maintain desired oxygen levels and prevent acetate accumulation. The final OD of the process was 260, and the volumetric yield of the H C fragment was 2065 mg/L, which reflects improvement by an order of magnitude. Purified H C fragments, produced by HCDC, exhibited typical biochemical and protective characteristics in mice. Taken together, the advancements achieved in this study promote large-scale production of the H C fragment in E. coli for use in anti-botulism vaccines.

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes.

The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of phar­maceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and, thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization in the SiMa lysate using a specific quantitative sandwich ELISA for its cleaved cellular target protein SNAP-25. The potencies of different pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accor­dance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤ 20) of next-generation equine and rabbit therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.