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The monogenetic trematode Gyrodactylus perforarus was previously reported to infect populations of the arrow goby, Clevelandia ios, from Bodega Bay, California. Clevelandia ios were sampled from Bodega Bay and nearby [...]

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.

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.

Antitoxin, the only licensed drug therapy for botulism, neutralizes circulating botulinum neurotoxin (BoNT). However, antitoxin is no longer effective when a critical amount of BoNT has already entered its target nerve cells. The outcome is a chronic phase of botulism that is characterized by prolonged paralysis. In this stage, blocking toxin activity within cells by next-generation intraneuronal anti-botulinum drugs (INABDs) may shorten the chronic phase of the disease and accelerate recovery. However, there is a lack of adequate animal models that simulate the chronic phase of botulism for evaluating the efficacy of INABDs. Herein, we report the development of a rabbit model for the chronic phase of botulism, induced by intoxication with a sublethal dose of BoNT. Spirometry monitoring enabled us to detect deviations from normal respiration and to quantitatively define the time to symptom onset and disease duration. A 0.85 rabbit intramuscular median lethal dose of BoNT/A elicited the most consistent and prolonged disease duration (mean = 11.8 days, relative standard deviation = 27.9%) that still enabled spontaneous recovery. Post-exposure treatment with antitoxin at various time points significantly shortened the disease duration, providing a proof of concept that the new model is adequate for evaluating novel therapeutics for botulism.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death ( p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

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 (LD 50 s) 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 LD 50 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC ® 1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

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., 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 © 2021 Dor, David, Dekel Jaoui, Schwartz, Baruchi, Torgeman, Ben David, Rosen, Tal, Rosner, Zichel and Diamant.)

Botulinum neurotoxin (BoNT) serotypes A, B and E are responsible for most cases of human botulism. The only approved therapy for botulism is antitoxin treatment administered to patients after symptom onset. However, a recent meta-analysis of antitoxin efficacy in human botulism cases over the past century concluded that a statistically significant reduction in mortality is associated with the use of type E and type A antitoxin, but not with type B antitoxin. Animal models could be highly valuable in studying postsymptom antitoxin efficacy (PSAE). However, the few attempts to evaluate PSAE in animals relied on subjective observations and showed ∼50% protection. Recently, we developed a novel spirometry model for the quantitative evaluation of PSAE in rabbits and used it to demonstrate full protection against BoNT/E. In the current study, a comparative evaluation of PSAE in botulism types A and B was conducted using this quantitative respiratory model. A lethal dose of each toxin induced a comparable course of disease both in terms of time to symptoms (TTS, 41.9±1.3 and 40.6±1.1 h, respectively) and of time to death (TTD, 71.3±3.1 and 66.3±1.7 h, respectively). However, in accordance with the differential serotypic PSAE observed in humans, postsymptom antitoxin treatment was fully effective only in BoNT/A-intoxicated rabbits. This serotypic divergence was reflected by a positive and statistically significant correlation between TTS and TTD in BoNT/A-intoxicated rabbits (r=0.91, P =0.0006), but not in those intoxicated with BoNT/B (r=0.06, P =0.88). The rabbit spirometry system might be useful in the evaluation toolkit of botulism therapeutics, including those under development and intended to act when antitoxin is no longer effective., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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 (LD 50 ) 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., (Copyright © 2018 American Society for Microbiology.)

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.

Monoclonal antibodies (MAbs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. Nevertheless, MAbs potency is still relatively low when compared to conventional polyclonal Ab preparations. Moreover, the efficacy of an individual neutralizing MAb may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. These limitations of individual neutralizing MAbs can be overcome by using oligoclonal combinations of several MAbs with different specificities to the target antigen. Studies conducted in our lab and by others show that such combined MAb preparation may present substantial synergy in its potency over the calculated additive potency of its individual MAb components. Moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. In this review, the synergistic neutralization properties of combined oligoclonal Ab preparations are described. The effect of Ab affinity, autologous Fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed.

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.

The receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (H(C)), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed in Escherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg H(C) per liter). The product was highly immunogenic, protecting mice from a challenge with 10(5) 50% lethal dose (LD(50)) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with HC increased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first time in vivo inhibition of BoNT/A intoxication by H(C)/A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of HC/A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P = 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of H(C)/A to cross-inhibit BoNT/E intoxication was evaluated. The administration of H(C)/A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P = 0.003). Furthermore, a combination of H(C)/A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD(50) of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by H(C)/A.

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.

Ligand-independent signals that are produced by the B-cell antigen receptor (BCR) confer an important positive selection checkpoint for immature B cells. Generation of inappropriate signals imposes developmental arrest of immature B cells, though the fate of these cells has not been investigated. Studies have shown that the lack of CD19 results in inappropriate signaling. In immunoglobulin transgenic mice, this inappropriate signaling impairs positive selection and stimulates receptor editing. Here, we studied the extent and significance of receptor editing in CD19-regulated positive selection of normal, nontransgenic B lymphopoiesis, using our bone marrow culture system. We found that the lack of CD19 resulted in elevated tonic signaling and impaired maturation, as revealed by surface marker expression and by functional assays. Immature CD19-/- B cells did not suppress RAG and underwent intensive receptor editing attempts in culture. Finally, in vivo analysis of light-chain isotype expression and Jkappa use in CD19-/- mice validated our in vitro observations. Our results suggest that CD19 has an important function in regulating positive selection and maturation of nontransgenic B-cell precursors and that receptor editing is an important salvage mechanism for immature B cells that fail positive selection.

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.