Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis

Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components o

Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain epitopes targeted in auto-immune encephalitis

Abstract Background Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal disease attributed to auto-antibodies against the N-methyl-D-aspartate receptor (NMDAR). Full recovery is possible if therapy is initiated early in the disease course. Detection of ANRE antibodies in the cerebrospinal fluid (CSF) is essential for diagnosis. The assays for ANRE-associated IgGs often rely on cells transiently transfected with NMDAR genes. A cell line that stably expresses pathogenic NMDAR epitopes could improve standardization of the assays and provide antigen that could be used in commercial solid state assay systems. Results We expressed the amino terminal domain (ATD) of the GluN1 NMDAR subunit (NR1) as a fusion protein on the outer plasma membrane of 293T cells, creating a stable cell population (293T-ATD) that is recognized by ANRE patient monoclonal antibodies in flow cytometry and immunofluorescence assays. The ATD fusion protein also contains a Myc tag and a 6XHIS tag, which provide functionality for immunoassays and antigen purification, and a TEV protease site, which allows the ATD domain to be specifically released from the cells in essentially pure form. ATD mobilized from the 293T ATD cell line maintained the pathogenic ANRE epitopes in ELISA binding assays. CSF (3/4) and sera (4/4) from ANRE patients also bound the 293T-ATD cell line, whereas normal CSF and sera did not. Conclusions The 293T-ATD cell line is potentially adaptable to a variety of formats to identify antibodies associated with ANRE, including cell-based and soluble antigen formats, and demonstrates a useful method to produce complex proteins for research, drug discovery, and clinical diagnosis

Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo

Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus–based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA) measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible

Trivalent Vaccine against Botulinum Toxin Serotypes A, B, and E That Can Be Administered by the Mucosal Route▿

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

Poliovirus (PV)-specific intestinal IgAs are important for cessation of PV shedding in the gastrointestinal tract following an acute infection with wild type or vaccine-derived PV strains. We sought to produce IgA monoclonal antibodies (mAbs) with PV neutralizing activity. We first performed de novo IgA discovery from primary human B cells using a hybridoma method that allows assessment of mAb binding and expression on the hybridoma surface: On-Cell mAb Screening (OCMS™). Six IgA1 mAbs were cloned by this method; three potently neutralized type 3 Sabin and wt PV strains. The hybridoma mAbs were heterogeneous, expressed in monomeric, dimeric, and aberrant forms. We also used recombinant methods to convert two high-potency anti-PV IgG mAbs into dimeric IgA1 and IgA2 mAbs. Isotype switching did not substantially change their neutralization activities. To purify the recombinant mAbs, Protein L binding was used, and one of the mAbs required a single amino acid substitution in its κ LC in order to enable protein L binding. Lastly, we used OCMS to assess IgA expression on the surface of hybridomas and transiently transfected, adherent cells. These studies have generated potent anti-PV IgA mAbs, for use in animal models, as well as additional tools for the discovery and production of human IgA mAbs

A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants

A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants