Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

ABSTRACT Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103 and 104 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

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In vitro and in vivo Assessment of Silver Nanoparticles Against Clostridium botulinum Type A Botulinum

Current Drug Discovery Technologies ◽

10.2174/1570163815666180403163946 ◽

2019 ◽

Vol 16 (1) ◽

pp. 113-119 ◽

Cited By ~ 1

Author(s):

Mohammad Aminianfar ◽

Siavash Parvardeh ◽

Mohsen Soleimani

Keyword(s):

Silver Nanoparticles ◽

Botulinum Toxin ◽

Clostridium Botulinum ◽

Lethal Dose ◽

Type A ◽

Positive Control ◽

Negative Control ◽

Laboratory Animals ◽

Control Group ◽

Nano Silver

Background: Clostridium botulinum causes botulism, a serious paralytic illness that results from the ingestion of a botulinum toxin. Because silver nanoparticle products exhibit strong antimicrobial activity, applications for silver nanoparticles in healthcare have expanded. Therefore, the objective of the current study was to assess a therapeutic strategy for the treatment of botulism toxicity using silver nanoparticles. Methods: A preliminary test was conducted using doses that produce illness in laboratory animals to determine the absolute lethal dose (LD100) of botulinum toxin type A (BoNT/A) in mice. Next, the test animals were divided into six groups containing six mice each. Groups I, II and III were the negative control (botulinum toxin only), positive control-1 (nano-silver only) and positive control-2 (no treatment), respectively. The remaining groups were allocated to the toxin that was supplemented with three nano-silver treatments. Results: The mortality rates of mice caused by BoNT/A significantly reduced in the treatment groups with different doses and injection intervals of nano-silver when compared to the negative control group. BoNT/A toxicity induced by intraperitoneal injection of the toxin of Clostridium botulinum causes rapid death while when coupled with nano-osilver results in delayed death in mice. Conclusion: These results, while open to future improvement, represent a preliminary step towards the satisfactory control of BoNT/A with the use of silver nanoparticles for human protection against this bioterrorism threat. Further study in this area can elucidate the underlying mechanism for detoxifying BoNT/A by silver nanoparticles.

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Development of Neutralizing Monoclonal Antibodies for Oncogenic Human Papillomavirus Types 31, 33, 45, 52, and 58

Clinical and Vaccine Immunology ◽

10.1128/cvi.00773-13 ◽

2014 ◽

Vol 21 (4) ◽

pp. 587-593 ◽

Cited By ~ 16

Author(s):

Martha J. Brown ◽

Hanna Seitz ◽

Victoria Towne ◽

Martin Müller ◽

Adam C. Finnefrock

Keyword(s):

Monoclonal Antibodies ◽

Human Papillomavirus ◽

Immune Responses ◽

Neutralizing Antibodies ◽

Cervical Cancers ◽

Hpv Types ◽

Oncogenic Hpv ◽

Neutralizing Epitopes ◽

Selection Of

ABSTRACTHuman papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic.

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Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

10.1101/2021.02.03.429355 ◽

2021 ◽

Author(s):

Carl Graham ◽

Jeffrey Seow ◽

Isabella Huettner ◽

Hataf Khan ◽

Neophytos Kouphou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Infectious Virus ◽

Antigenic Drift ◽

Host Cells ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

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Airway Delivery of Anti-influenza Monoclonal Antibodies Results in Enhanced Antiviral Activities and Enables Broad-Coverage Combination Therapies

Journal of Virology ◽

10.1128/jvi.00052-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Adam Vigil ◽

Natalia Frias-Staheli ◽

Teresa Carabeo ◽

Michael Wittekind

Keyword(s):

Monoclonal Antibodies ◽

Therapeutic Approach ◽

Neutralizing Antibodies ◽

Lethal Dose ◽

Passive Immunization ◽

Cost Effective ◽

Effector Function ◽

Influenza Viruses ◽

Apical Side ◽

Promising Therapeutic Approach

ABSTRACT Effective and reliable anti-influenza treatments are acutely needed and passive immunizations using broadly neutralizing anti-influenza monoclonal antibodies (bNAbs) are a promising emerging approach. Because influenza infections are initiated in and localized to the pulmonary tract, and newly formed viral particles egress from the apical side of the lung epithelium, we compared the effectiveness of hemagglutinin (HA) stalk-binding bNAbs administered through the airway (intranasal or via nebulization) versus the systemic route (intraperitoneal or intravenous). Airway deliveries of various bNAbs were 10- to 50-fold more effective than systemic deliveries of the same bNAbs in treating H1N1, H3N2, B/Victoria-, and B/Yamagata-lineage influenza viral infections in mouse models. The potency of airway-delivered anti-HA bNAbs was highly dependent on antiviral neutralization activity, with little dependence on the effector function of the antibody. In contrast, the effectiveness of systemically delivered anti-HA bNAbs was not dependent on antiviral neutralization, but critically dependent on antibody effector functions. Concurrent administration of a neutralizing/effector function-positive bNAb via the airway and systemic routes showed increased effectiveness. The small amount of airway-delivered bNAbs needed for effective influenza treatment creates the opportunity to combine potent bNAbs with different anti-influenza specificities to generate a cost-effective antiviral therapy that provides broad coverage against all circulating influenza strains infecting humans. A 3 mg/kg dose of the novel triple antibody combination CF-404 (i.e., 1 mg/kg of each component bNAb) delivered to the airway was shown to effectively prevent weight loss and death in mice challenged with ten 50% lethal dose (LD50) inoculums of either H1N1, H3N2, B/Victoria-lineage, or B/Yamagata-lineage influenza viruses. IMPORTANCE Influenza causes widespread illness in humans and can result in morbidity and death, especially in the very young and elderly populations. Because influenza vaccination can be poorly effective some years, and the immune systems of the most susceptible populations are often compromised, passive immunization treatments using broadly neutralizing antibodies is a promising therapeutic approach. However, large amounts of a single antibody are required for effectiveness when delivered through systemic administration (typically intravenous infusion), precluding the feasible dosing of antibody combinations via this route. The significance of our research is the demonstration that effective therapeutic treatments of multiple relevant influenza types (H1N1, H3N2, and B) can be achieved by airway administration of a single combination of relatively small amounts of three anti-influenza antibodies. This advance exploits the discovery that airway delivery is a more potent way of administering anti-influenza antibodies compared to systemic delivery, making this a feasible and cost-effective therapeutic approach.

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Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

Applied and Environmental Microbiology ◽

10.1128/aem.72.2.1231-1238.2006 ◽

2006 ◽

Vol 72 (2) ◽

pp. 1231-1238 ◽

Cited By ~ 110

Author(s):

Shashi K. Sharma ◽

Joseph. L. Ferreira ◽

Brian S. Eblen ◽

Richard C. Whiting

Keyword(s):

Immunosorbent Assay ◽

Clostridium Botulinum ◽

Botulinum Neurotoxin ◽

Large Scale ◽

Lethal Dose ◽

Meat Products ◽

Type A ◽

Detection Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Botulinum Neurotoxins

ABSTRACT An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

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Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies

Vaccines ◽

10.3390/vaccines9020135 ◽

2021 ◽

Vol 9 (2) ◽

pp. 135

Author(s):

Shujie Wang ◽

Chunsheng Wang ◽

Xiao Ren ◽

Wenjiao Xue ◽

Haijuan He ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

3D Structure ◽

Surface Region ◽

Neutralization Titer ◽

Random Peptide ◽

Hexon Protein ◽

Potential Applications ◽

Alignment Analysis ◽

Neutralizing Epitopes

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

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Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

10.1101/2020.07.21.214759 ◽

2020 ◽

Cited By ~ 19

Author(s):

Yiska Weisblum ◽

Fabian Schmidt ◽

Fengwen Zhang ◽

Justin DaSilva ◽

Daniel Poston ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Receptor Binding Domain ◽

Reporter Virus ◽

Low Frequencies ◽

Protein Variants ◽

Prior Infection ◽

Neutralizing Epitopes

AbstractNeutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

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Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-378 ◽

2015 ◽

Vol 78 (8) ◽

pp. 1506-1511

Author(s):

GUY E. SKINNER ◽

GREGORY J. FLEISCHMAN ◽

FRAN BALSTER ◽

KARL REINEKE ◽

N. RUKMA REDDY ◽

...

Keyword(s):

Clostridium Botulinum ◽

Apple Juice ◽

Lethal Dose ◽

Type A ◽

The United States ◽

Mouse Bioassay ◽

Potential Threat ◽

Complete Inactivation ◽

Inverted Orientation ◽

Botulinum Type

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼106 mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 106 MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 106 MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 105 MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 106 MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 104 MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

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Competitive Inhibition between Different Clostridium botulinum Types and Strains

Journal of Food Protection ◽

10.4315/0362-028x-67.12.2682 ◽

2004 ◽

Vol 67 (12) ◽

pp. 2682-2687 ◽

Cited By ~ 4

Author(s):

M. W. EKLUND ◽

F. T. POYSKY ◽

M. E. PETERSON ◽

R. N. PARANJPYE ◽

G. A. PELROY

Keyword(s):

Clostridium Botulinum ◽

Competitive Inhibition ◽

Type A ◽

Toxin Production ◽

Inhibitory Effect ◽

Potential Effect ◽

Type B ◽

Clostridium Sporogenes ◽

Different Strains ◽

Production Strain

Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C. botulinum were tested against each other for the purpose of selecting a mixture of compatible C. botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents. All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C. sporogenes. When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited. Nonproteolytic strains of C. botulinum also produced bacteriocin-like agents against each other. Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C. botulinum and C. sporogenes. EF4, however, was not inhibitory to the nontoxigenic E-like strains. When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production. Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30°C. On the basis of the production of these bacteriocin-like agents by different strains of C. botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.

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Isolation of Porcine Reproductive and Respiratory Syndrome Virus GP5-Specific, Neutralizing Monoclonal Antibodies From Hyperimmune Sows

Frontiers in Immunology ◽

10.3389/fimmu.2021.638493 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jordan E. Young ◽

Cheryl M. T. Dvorak ◽

Simon P. Graham ◽

Michael P. Murtaugh

Keyword(s):

Monoclonal Antibodies ◽

B Cells ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Growing Pigs ◽

Respiratory Syndrome Virus ◽

Broadly Neutralizing Antibodies ◽

Complete Protection ◽

Broadly Neutralizing Antibody ◽

Neutralizing Epitopes

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease which impacts the pig industry worldwide. The disease is caused by PRRS viruses (PRRSV-1 and -2) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in growing pigs. Current PRRSV vaccines provide limited protection; only providing complete protection against closely related strains. The development of improved PRRSV vaccines would benefit from an increased understanding of epitopes relevant to protection, including those recognized by antibodies which possess the ability to neutralize distantly related strains. In this work, a reverse vaccinology approach was taken; starting first with pigs known to have a broadly neutralizing antibody response and then investigating the responsible B cells/antibodies through the isolation of PRRSV neutralizing monoclonal antibodies (mAbs). PBMCs were harvested from pigs sequentially exposed to a modified-live PRRSV-2 vaccine as well as divergent PRRSV-2 field isolates. Memory B cells were immortalized and a total of 5 PRRSV-specific B-cell populations were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1 isolates. Antibodies against GP5 protein, commonly thought to possess a dominant PRRSV neutralizing epitope, were found to be highly abundant, as four out of five B cells populations were GP5 specific. One of the GP5-specific mAbs was shown to be neutralizing but this was only observed against homologous and not heterologous PRRSV strains. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of broadly neutralizing antibodies against other porcine pathogens.

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