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Article ; Online: Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples

Efi Makdasi / Orly Laskar / Itai Glinert / Ron Alcalay / Adva Mechaly / Haim Levy

Pathogens, Vol 9, Iss 3, p

2020 Volume 164

Abstract: Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both ... ...

Abstract	: Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both challenging and time-consuming. This is due to the high homology with other Bacillus species as well as the complex composition of environmental samples, which further impedes assay sensitivity. Previously, we showed that a short incubation of B.anthracis spores in a defined growth medium results in rapid germination, bacterial growth, and secretion of toxins, including protective antigen. In this work, we tested whether coupling the incubation process to a newly developed immune-assay will enable the detection of secreted toxins as markers for the presence of spores in environmental samples. The new immune assay is a flow cytometry-based multiplex that simultaneously detects a protective antigen, lethal factor, and edema factor. Our combined assay detects 1 × 10 3 −1 × 10 4 /mL spores after a 2 h incubation followed by the ~80 min immune-multiplex detection. Extending the incubation step to 5 h increased assay sensitivity to 1 × 10 2 /mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent B. anthracis spores. There was no substantial influence of contaminants derived from real environmental samples on the performance of the assay compared to clean samples, which allow the unequivocal detection of 3 × 10 3 /mL and 3 × 10 2 /mL spores following 2 and 5 hour’s incubation, respectively. Overall, we propose this method as a rapid, sensitive, and specific procedure for the identification of B. anthracis spores in environmental samples.
Keywords	bacillus anthracis ; anthrax ; environment samples ; multiplex ; protective antigen ; lethal factor ; edema factor ; Medicine ; R
Subject code	333
Language	English
Publishing date	2020-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Characterization of antibody-antigen interactions using biolayer interferometry

Tal Noy-Porat / Ron Alcalay / Adva Mechaly / Eldar Peretz / Efi Makdasi / Ronit Rosenfeld / Ohad Mazor

STAR Protocols, Vol 2, Iss 4, Pp 100836- (2021)

2021

Abstract: Summary: This protocol describes the use of a biolayer interferometry platform for assessing antibody-antigen interactions. The protocol focuses on affinity determination and epitope binning, although the system can be utilized for measuring any protein- ... ...

Abstract	Summary: This protocol describes the use of a biolayer interferometry platform for assessing antibody-antigen interactions. The protocol focuses on affinity determination and epitope binning, although the system can be utilized for measuring any protein-protein interaction. Readings are collected in real time, allowing the use of unlabeled molecules, and data can thus be obtained in a fast and easy manner. Experiments should be carefully designed, taking into consideration the tested interaction, available sensors, and suitable controls.For complete details on the use and execution of this protocol, please refer to Noy-Porat et al. (2021).
Keywords	Immunology ; Antibody ; Protein Biochemistry ; Science (General) ; Q1-390
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Inhibition of Francisella tularensis phagocytosis using a novel anti-LPS scFv antibody fragment

Adva Mechaly / Uri Elia / Ron Alcalay / Hila Cohen / Eyal Epstein / Ofer Cohen / Ohad Mazor

Scientific Reports, Vol 9, Iss 1, Pp 1-

2019 Volume 9

Abstract: Abstract Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting ... ...

Abstract	Abstract Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.
Keywords	Medicine ; R ; Science ; Q
Subject code	572
Language	English
Publishing date	2019-08-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Immunodominant Linear B-Cell Epitopes of SARS-CoV-2 Spike, Identified by Sera from K18-hACE2 Mice Infected with the WT or Variant Viruses

Yinon Levy / Ron Alcalay / Anat Zvi / Efi Makdasi / Eldar Peretz / Tal Noy-Porat / Theodor Chitlaru / Michal Mandelboim / Ohad Mazor / Ronit Rosenfeld

Vaccines, Vol 10, Iss 251, p

2022 Volume 251

Abstract: SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of ... ...

Abstract	SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of SARS-CoV-2 proteins in general, and that of the spike protein in particular, may contribute to the development of sensitive diagnostic tools and identification of vaccine` candidate targets. In the current study, the anti-viral antibody response in transgenic K18-hACE-2 mice was examined by implementing an immunodominant epitope mapping approach of the SARS-CoV-2 spike. Serum samples for probing an epitope array covering the entire spike protein were collected from mice following infection with the original SARS-CoV-2 strain as well as the B.1.1.7 Alpha and B.1.351 Beta genetic variants of concern. The analysis resulted in distinction of six linear epitopes common to the humoral response against all virus variants inspected at a frequency of more than 20% of the serum samples. Finally, the universality of the response was probed by cross-protective in vitro experiments using plaque-reducing neutralization tests. The data presented here has important implications for prediction of the efficacy of immune countermeasures against emerging SARS-CoV-2 variants.
Keywords	COVID-19 ; SARS-CoV-2 ; epitope mapping ; linear epitopes ; K18-hACE2 ; Medicine ; R
Subject code	570
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Prolonged Protective Immunity Induced by Mild SARS-CoV-2 Infection of K18-hACE2 Mice

Liat Bar-On / Moshe Aftalion / Efi Makdasi / David Gur / Ron Alcalay / Hila Cohen / Adi Beth-Din / Ronit Rosenfeld / Hagit Achdout / Erez Bar-Haim / Reut Falach / Theodor Chitlaru / Ofer Cohen

Vaccines, Vol 10, Iss 613, p

2022 Volume 613

Abstract: Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show ... ...

Abstract	Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.
Keywords	SARS-CoV-2 ; COVID-19 ; K18-hACE2 mice ; mild infection ; Medicine ; R
Language	English
Publishing date	2022-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Novel Phage Display-Derived Anti-Abrin Antibodies Confer Post-Exposure Protection against Abrin Intoxication

Adva Mechaly / Ron Alcalay / Tal Noy-Porat / Eyal Epstein / Yoav Gal / Ohad Mazor

Toxins, Vol 10, Iss 2, p

2018 Volume 80

Abstract: Abrin toxin is a type 2 ribosome inactivating glycoprotein isolated from the seeds of Abrus precatorius (jequirity pea). Owing to its high toxicity, relative ease of purification and accessibility, it is considered a biological threat agent. To date, ... ...

Abstract	Abrin toxin is a type 2 ribosome inactivating glycoprotein isolated from the seeds of Abrus precatorius (jequirity pea). Owing to its high toxicity, relative ease of purification and accessibility, it is considered a biological threat agent. To date, there is no effective post-exposure treatment for abrin poisoning and passive immunization remains the most effective therapy. However, the effectiveness of anti-abrin monoclonal antibodies for post-exposure therapy following abrin intoxication has not been demonstrated. The aim of this study was to isolate high affinity anti-abrin antibodies that possess potent toxin-neutralization capabilities. An immune scFv phage-display library was constructed from an abrin-immunized rabbit and a panel of antibodies (six directed against the A subunit of abrin and four against the B subunit) was isolated and expressed as scFv-Fc antibodies. By pair-wise analysis, we found that these antibodies target five distinct epitopes on the surface of abrin and that antibodies against all these sites can bind the toxin simultaneously. Several of these antibodies (namely, RB9, RB10, RB28 and RB30) conferred high protection against pulmonary intoxication of mice, when administered six hours post exposure to a lethal dose of abrin. The data presented in this study demonstrate for the first time the efficacy of monoclonal antibodies in treatment of mice after pulmonary intoxication with abrin and promote the use of these antibodies, one or several, for post-exposure treatment of abrin intoxication.
Keywords	abrin ; monoclonal antibodies ; phage-display ; rabbit ; recombinant antibodies ; Medicine ; R
Subject code	616
Language	English
Publishing date	2018-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Extended therapeutic window for post-exposure treatment of ricin intoxication conferred by the use of high-affinity antibodies

Noy-Porat, Tal / Ron Alcalay / Eyal Epstein / Tamar Sabo / Chanoch Kronman / Ohad Mazor

Toxicon. 2017 Mar. 01, v. 127

2017

Abstract: The plant toxin ricin is considered a potential bioterror agent against which there is no available antidote. To date, neutralizing antibodies are the most promising post-exposure treatment for ricin intoxication, yet so far they were shown to be ... ...

Abstract	The plant toxin ricin is considered a potential bioterror agent against which there is no available antidote. To date, neutralizing antibodies are the most promising post-exposure treatment for ricin intoxication, yet so far they were shown to be effective only when given within several hours post exposure. As part of an ongoing effort to develop efficient ricin-countermeasures, we tested whether high-affinity antibodies that were previously isolated from immunized non-human primates, may confer effective post-exposure therapy for ricin-intoxicated mice treated at late time-points after exposure. While each antibody is capable of providing high protection rate by itself, a formulation consisting of three neutralizing antibodies that target different epitopes was tested to provide therapeutic coverage against different variants of the malicious pathogen. Indeed, the tri-antibody based cocktail was highly effective, its administration resulting in very high survival rates (>70%) when animals were treated as late as 48Â h post exposure and significant protection (>30%) even at 72Â h. This study establishes for the first time that anti-ricin antibodies can serve as a highly effective antidote at such late time-points after exposure. From the clinical point of view, the extended therapeutic window documented here is of high importance allowing adequate time to accurately identify the causative agent and may permit initiation of life-saving treatment with these antibodies even after the onset of clinical signs.
Keywords	Primates ; antidotes ; epitopes ; mice ; neutralization ; neutralizing antibodies ; pathogens ; poisoning ; ricin ; survival rate ; therapeutics
Language	English
Dates of publication	2017-0301
Size	p. 100-105.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	204479-1
ISSN	1879-3150 ; 0041-0101
ISSN (online)	1879-3150
ISSN	0041-0101
DOI	10.1016/j.toxicon.2017.01.009
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

Tal Noy-Porat / Ronit Rosenfeld / Naomi Ariel / Eyal Epstein / Ron Alcalay / Anat Zvi / Chanoch Kronman / Arie Ordentlich / Ohad Mazor

Toxins, Vol 8, Iss 3, p

2016 Volume 64

Abstract: Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ... ...

Abstract	Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.
Keywords	ricin ; antibody ; neutralization ; affinity ; immunization ; non-human primates ; Medicine ; R
Language	English
Publishing date	2016-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody

Nature Communications, Vol 12, Iss 1, Pp 1-

2021 Volume 9

Abstract: Here, using the K18-hACE2 transgenic mice model, the authors report the in vivo efficacy of a fully human neutralizing antibody against SARS-CoV-2 and show that when administered before or up to 3 days post infection, treated mice do not exhibit disease ... ...

Abstract	Here, using the K18-hACE2 transgenic mice model, the authors report the in vivo efficacy of a fully human neutralizing antibody against SARS-CoV-2 and show that when administered before or up to 3 days post infection, treated mice do not exhibit disease symptoms while 80% of control animals succumb to the infection.
Keywords	Science ; Q
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

iScience, Vol 24, Iss 5, Pp 102479- (2021)

2021

Abstract: Summary: Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal ... ...

Abstract	Summary: Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal antibodies (mAbs), targeting several distinct epitopes for circumventing therapy escape mutants. Previously, we reported efficient human therapeutic mAbs recognizing epitopes on the spike receptor-binding domain (RBD) of SARS-CoV-2. Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N-glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late post-exposure, demonstrating the high potential of the mAbs for therapy of SARS-CoV-2 infection.
Keywords	Molecular biology ; Immunology ; Virology ; Science ; Q
Subject code	570
Language	English
Publishing date	2021-05-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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