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Article: Development and Validation of a Plaque Assay to Determine the Titer of a Recombinant Live-Attenuated Viral Vaccine for SARS-CoV-2.

Toister, Einat / Cherry, Lilach / Lupu, Edith / Monash, Arik / Dor, Eyal / Levin, Lilach / Girshengorn, Meni / Natan, Niva / Chapman, Shira / Shmaya, Shlomo / Epstein, Eyal / Adar, Yaakov / Zichel, Ran / Ophir, Yakir / Diamant, Eran

Vaccines

2024 Volume 12, Issue 4

Abstract: 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 ... ...

Abstract	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
Language	English
Publishing date	2024-04-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2703319-3
ISSN	2076-393X
ISSN	2076-393X
DOI	10.3390/vaccines12040374
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Article ; Online: High Cell Density Cultivation Process for the Expression of Botulinum Neurotoxin a Receptor Binding Domain.

Ben David, Alon / Papir, Yoel / Hazan, Ophir / Redelman, Moses / Diamant, Eran / Barnea, Ada / Torgeman, Amram / Zichel, Ran

Toxins

2022 Volume 14, Issue 4

Abstract: The receptor-binding domain of botulinum neurotoxin ( ... ...

Abstract	The receptor-binding domain of botulinum neurotoxin (H
MeSH term(s)	Animals ; Botulinum Toxins, Type A/metabolism ; Botulism/prevention & control ; Cell Count ; Culture Media/metabolism ; Escherichia coli ; Fermentation ; Mice ; Recombinant Proteins/metabolism
Chemical Substances	Culture Media ; Recombinant Proteins ; Botulinum Toxins, Type A (EC 3.4.24.69)
Language	English
Publishing date	2022-04-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2518395-3
ISSN	2072-6651 ; 2072-6651
ISSN (online)	2072-6651
ISSN	2072-6651
DOI	10.3390/toxins14040281
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Article ; Online: Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.

Ben David, Alon / Barnea, Ada / Diamant, Eran / Dor, Eyal / Schwartz, Arieh / Torgeman, Amram / Zichel, Ran

International journal of molecular sciences

2021 Volume 22, Issue 16

Abstract: 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 ... ...

Abstract	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 (
MeSH term(s)	Animals ; Aurintricarboxylic Acid/pharmacology ; Botulinum Toxins/toxicity ; Botulinum Toxins, Type A/toxicity ; Botulism/drug therapy ; Botulism/genetics ; Botulism/metabolism ; Membrane Glycoproteins/antagonists & inhibitors ; Membrane Glycoproteins/genetics ; Membrane Glycoproteins/metabolism ; Mice ; Nerve Tissue Proteins/antagonists & inhibitors ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; beta-Galactosidase/genetics ; beta-Galactosidase/metabolism
Chemical Substances	Membrane Glycoproteins ; Nerve Tissue Proteins ; Recombinant Fusion Proteins ; Sv2a protein, mouse ; Aurintricarboxylic Acid (4431-00-9) ; beta-Galactosidase (EC 3.2.1.23) ; Botulinum Toxins (EC 3.4.24.69) ; Botulinum Toxins, Type A (EC 3.4.24.69) ; botulinum toxin type E (T579M564JY)
Language	English
Publishing date	2021-08-09
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms22168577
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Article ; Online: A Novel Running Wheel Mouse Model for Botulism and Its Use for the Evaluation of Postsymptom Antitoxin Efficacy.

Schwartz, Arieh / Ben David, Alon / Hotoveli, Mordechai / Dor, Eyal / Diamant, Eran / Vivyorka, Arik / Rosen, Osnat / Torgeman, Amram / Zichel, Ran

Antimicrobial agents and chemotherapy

2021 Volume 65, Issue 8, Page(s) e0042121

Abstract: 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 ... ...

Abstract	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
MeSH term(s)	Animals ; Antitoxins/therapeutic use ; Botulinum Toxins, Type A ; Botulism/diagnosis ; Botulism/drug therapy ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Humans ; Mice ; Serogroup
Chemical Substances	Antitoxins ; Botulinum Toxins, Type A (EC 3.4.24.69)
Language	English
Publishing date	2021-07-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	217602-6
ISSN	1098-6596 ; 0066-4804
ISSN (online)	1098-6596
ISSN	0066-4804
DOI	10.1128/AAC.00421-21
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Article ; Online: A Rabbit Model for the Evaluation of Drugs for Treating the Chronic Phase of Botulism.

Torgeman, Amram / Diamant, Eran / Dor, Eyal / Schwartz, Arieh / Baruchi, Tzadok / Ben David, Alon / Zichel, Ran

Toxins

2021 Volume 13, Issue 10

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2021-09-24
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2518395-3
ISSN	2072-6651 ; 2072-6651
ISSN (online)	2072-6651
ISSN	2072-6651
DOI	10.3390/toxins13100679
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Article: Immunologic and Protective Properties of Subunit- vs. Whole Toxoid-Derived Anti-Botulinum Equine Antitoxin.

Ben David, Alon / Barnea, Ada / Torgeman, Amram / Diamant, Eran / Dor, Eyal / Schwartz, Arieh / Rosen, Osnat / Caspi, Noa / Saraf, Miki / Lerer, Elad / Adar, Yaakov / Lupo, Edith / Toister, Einat / Zichel, Ran

Vaccines

2022 Volume 10, Issue 9

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2022-09-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2703319-3
ISSN	2076-393X
ISSN	2076-393X
DOI	10.3390/vaccines10091522
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Article ; Online: Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope.

Mechaly, Adva / Diamant, Eran / Alcalay, Ron / Ben David, Alon / Dor, Eyal / Torgeman, Amram / Barnea, Ada / Girshengorn, Meni / Levin, Lilach / Epstein, Eyal / Tennenhouse, Ariel / Fleishman, Sarel J / Zichel, Ran / Mazor, Ohad

Antibodies (Basel, Switzerland)

2022 Volume 11, Issue 1

Abstract: 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- ...

Abstract	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.
Language	English
Publishing date	2022-03-16
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2661514-9
ISSN	2073-4468 ; 2073-4468
ISSN (online)	2073-4468
ISSN	2073-4468
DOI	10.3390/antib11010021
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Article ; Online: Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs.

Diamant, Eran / Torgeman, Amram / Ozeri, Eyal / Zichel, Ran

Toxins

2015 Volume 7, Issue 6, Page(s) 1854–1881

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Monoclonal/pharmacology ; Antibodies, Monoclonal/therapeutic use ; Antibodies, Neutralizing/pharmacology ; Antibodies, Neutralizing/therapeutic use ; Drug Synergism ; Humans
Chemical Substances	Antibodies, Monoclonal ; Antibodies, Neutralizing
Keywords	covid19
Language	English
Publishing date	2015-05-29
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2518395-3
ISSN	2072-6651 ; 2072-6651
ISSN (online)	2072-6651
ISSN	2072-6651
DOI	10.3390/toxins7061854
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Article ; Online: Development and Validation of an Innovative Analytical Approach for the Quantitation of Tris(Hydroxymethyl)Aminomethane (TRIS) in Pharmaceutical Formulations by Liquid Chromatography Tandem Mass Spectrometry.

Madmon, Moran / Shamai Yamin, Tamar / Pitel, Shani / Belay, Chen / Segula, Yaniv / Toister, Einat / Hindi, Ariel / Cherry, Lilach / Ophir, Yakir / Zichel, Ran / Mimran, Avishai / Diamant, Eran / Weissberg, Avi

Molecules (Basel, Switzerland)

2022 Volume 28, Issue 1

Abstract: 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( ... ...

Abstract	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.
MeSH term(s)	Humans ; COVID-19 Vaccines ; Tromethamine/chemistry ; Tandem Mass Spectrometry/methods ; Chromatography, High Pressure Liquid/methods ; Drug Compounding ; COVID-19/prevention & control ; SARS-CoV-2 ; Chromatography, Liquid
Chemical Substances	rVSV-deltaG-spike COVID-19 vaccine ; COVID-19 Vaccines ; Tromethamine (023C2WHX2V)
Language	English
Publishing date	2022-12-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1413402-0
ISSN	1420-3049 ; 1431-5165 ; 1420-3049
ISSN (online)	1420-3049
ISSN	1431-5165 ; 1420-3049
DOI	10.3390/molecules28010073
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Article: A Rabbit Model for Prolonged Continuous Intravenous Infusion Via a Peripherally Inserted Central Catheter.

Dor, Eyal / David, Tseela / Dekel Jaoui, Hani / Schwartz, Arieh / Baruchi, Tzadok / Torgeman, Amram / Ben David, Alon / Rosen, Osnat / Tal, Arnon / Rosner, Amir / Zichel, Ran / Diamant, Eran

Frontiers in pharmacology

2021 Volume 12, Page(s) 637792

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2021-04-08
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587355-6
ISSN	1663-9812
ISSN	1663-9812
DOI	10.3389/fphar.2021.637792
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