Eran Diamant

Bio: Eran Diamant is an academic researcher from Israel Institute for Biological Research. The author has contributed to research in topics: Antitoxin & Botulism. The author has an hindex of 6, co-authored 17 publications receiving 94 citations.

Monoclonal Antibody Combinations that Present Synergistic Neutralizing Activity: A Platform for Next-Generation Anti-Toxin Drugs.

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

Evaluating the Synergistic Neutralizing Effect of Anti-Botulinum Oligoclonal Antibody Preparations

Abstract: 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.

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries.

Abstract: 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 06 to 55 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

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

Abstract: 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.

Role of Homologous Fc Fragment in the Potency and Efficacy of Anti-Botulinum Antibody Preparations.

Abstract: 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.

Peptides, Antibodies, Peptide Antibodies and More.

Abstract: The applications of peptides and antibodies to multiple targets have emerged as powerful tools in research, diagnostics, vaccine development, and therapeutics. Antibodies are unique since they, in theory, can be directed to any desired target, which illustrates their versatile nature and broad spectrum of use as illustrated by numerous applications of peptide antibodies. In recent years, due to the inherent limitations such as size and physical properties of antibodies, it has been attempted to generate new molecular compounds with equally high specificity and affinity, albeit with relatively low success. Based on this, peptides, antibodies, and peptide antibodies have established their importance and remain crucial reagents in molecular biology.

Tables of Toxicity of Botulinum and Tetanus Neurotoxins.

Abstract: Tetanus and botulinum neurotoxins are the most poisonous substances known, so much so as to be considered for a possible terrorist use. At the same time, botulinum neurotoxin type A1 is successfully used to treat a variety of human syndromes characterized by hyperactive cholinergic nerve terminals. The extreme toxicity of these neurotoxins is due to their neurospecificity and to their metalloprotease activity, which results in the deadly paralysis of tetanus and botulism. Recently, many novel botulinum neurotoxins and some botulinum-like toxins have been discovered. This large number of toxins differs in terms of toxicity and biological activity, providing a potential goldmine for novel therapeutics and for new molecular tools to dissect vesicular trafficking, fusion, and exocytosis. The scattered data on toxicity present in the literature require a systematic organization to be usable by scientists and clinicians. We have assembled here the data available in the literature on the toxicity of these toxins in different animal species. The internal comparison of these data provides insights on the biological activity of these toxins.

Cross-Reactive Antibodies Prevent the Lethal Effects of Staphylococcus Aureus Superantigens

Abstract: The exotoxins produced by Staphylococcus aureus, staphylococcal enterotoxins (SE) A-E and toxic shock syndrome toxin (TSST)-1, which are associated with serious diseases, including food poisoning and toxic shock syndrome, are termed superantigens (SAgs). To examine whether common antigenic epitopes were present and whether vaccination with 1 bacterial SAg could protect against challenge with a different SE or TSST-1, mice were vaccinated with SEA, SEB, SEC1, or TSST-1 individually or in combination. Mice injected with a single toxin developed high antibody titers against other SAgs. Marked improvement in survival was observed when immunized mice were challenged with a heterologous toxin. Mice vaccinated with a mixture of toxins were fully protected against 1 or multiple toxin challenges, indicating no interference effects of multivalent vaccinations. More importantly, higher titers were found against each SAg with the multivalent vaccination than with injection with a single SAg. Thus, immunizations with 1 SAg can induce cross-protective antibodies to heterologous SAgs, and multicomponent vaccination can enhance antibody responses against each bacterial SAg.

Proteomic Methods of Detection and Quantification of Protein Toxins.

Abstract: Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins, Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis, Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album. The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents.