ABSTRACT
Botulinum neurotoxins are some of the most potent natural toxins known; they cause flaccid paralysis by inhibiting synaptic vesicle release. Some serotypes, notably serotype A and B, can cause persistent paralysis lasting for several months. Because of their potency and persistence, botulinum neurotoxins are now used to manage several clinical conditions, and there is interest in expanding their clinical applications using engineered toxins with novel substrate specificities. It will also be beneficial to engineer toxins with tunable persistence. We have investigated the potential use of small-molecule proteolysis-targeting chimeras (PROTACs) to vary the persistence of modified recombinant botulinum neurotoxins. We also describe a complementary approach that has potential relevance for botulism treatment. This second approach uses a camelid heavy chain antibody directed against botulinum neurotoxin that is modified to bind the PROTAC. These strategies provide proof of principle for the use of two different approaches to fine tune the persistence of botulinum neurotoxins by selectively targeting their catalytic light chains for proteasomal degradation.
Subject(s)
Botulinum Toxins , Proteolysis , Botulinum Toxins/chemistry , Botulinum Toxins/metabolism , Humans , Animals , Proteasome Endopeptidase Complex/metabolism , Proteolysis Targeting ChimeraABSTRACT
Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.
Subject(s)
Lipoproteins , Lyme Disease , Single-Domain Antibodies , Animals , Dogs , Humans , Lyme Disease Vaccines , Epitopes , Antibodies, Bacterial , Bacterial Vaccines , Bacterial Outer Membrane Proteins , Lyme Disease/prevention & control , Antigens, Surface , Antibodies, MonoclonalABSTRACT
Single-domain antibodies (sdAbs) derived from Camelidae heavy-chain-only antibodies (also called nanobodies or VHHs) have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins. Enteric PK data obtained from in vitro incubation studies were consistent with similar incubations performed in vivo in rabbit surgical gut loops. These findings have broad implications for enteric use of VHH-based agents, particularly VHH fusion proteins.
Subject(s)
Camelids, New World , Single-Domain Antibodies , Animals , Humans , Rabbits , Swine , Immunoglobulin Heavy Chains , Antibodies, Monoclonal , Amino Acid Sequence , Peptide HydrolasesABSTRACT
The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here we report on the development of mRNA-LNPs encoding camelid-derived VHH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.
Subject(s)
Clostridioides difficile , Single-Domain Antibodies , Swine , Animals , Mice , RNA , Half-Life , Antibodies , RNA, MessengerABSTRACT
Schistosomiasis is a globally prevalent, debilitating disease that is poorly controlled by chemotherapy and for which no vaccine exists. While partial resistance in people may develop over time with repeated infections and treatments, some animals, including the brown rat (Rattus norvegicus), are only semi-permissive and have natural protection. To understand the basis of this protection, we explored the nature of the immune response in the brown rat to infection by Schistosoma mansoni. Infection leads to production of IgG to parasite glycoproteins with complex-type N-glycans that contain a non-mammalian-type modification by core α2-Xylose and core α3-Fucose (core Xyl/Fuc). These epitopes are expressed on the surfaces of schistosomula and adult worms. Importantly, IgG to these epitopes can kill schistosomula by a complement-dependent process in vitro. Additionally, sera from both infected rhesus monkey and infected brown rat were capable of killing schistosomula in a manner inhibited by glycopeptides containing core Xyl/Fuc. These results demonstrate that protective antibodies to schistosome infections in brown rats and rhesus monkeys include IgG responses to the core Xyl/Fuc epitopes in surface-expressed N-glycans, and raise the potential of novel glyco-based vaccines that might be developed to combat this disease.
ABSTRACT
Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT3EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT3EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT3EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT3EcT as a platform for the treatment of gastrointestinal-based diseases.
Subject(s)
Colitis , Single-Domain Antibodies , Animals , Mice , Escherichia coli , Colitis/chemically induced , Colitis/therapy , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.
Subject(s)
Botulinum Toxins, Type A , Synaptic Vesicles , Humans , Synaptic Vesicles/metabolism , Botulinum Toxins, Type A/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Protein BindingABSTRACT
While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.
Subject(s)
Botulinum Toxins , Single-Domain Antibodies , Amino Acids/chemistry , Botulinum Toxins/immunology , Genetic Code , Saccharomyces cerevisiae/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/isolation & purificationABSTRACT
Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.
Subject(s)
Bacterial Toxins , Cryptosporidiosis , Cryptosporidium , Enteropathogenic Escherichia coli , Probiotics , Single-Domain Antibodies , Humans , Antigens, Surface , Congo Red , Flagellin , Type III Secretion Systems , Virulence Factors/geneticsABSTRACT
Single domain antibodies (sdAbs), also called nanobodies, have substantial biophysical advantages over conventional antibodies and are increasingly being employed as components of immunotherapeutic agents. One particularly favorable property is the ability to link different sdAbs into heteromultimers. This feature allows production of single molecules capable of simultaneously targeting more than one antigen. In addition, cooperative binding of multiple linked sdAbs to non-overlapping epitopes on the same target can produce synergistic improvements in target affinity, variant specificity, and in vivo potencies. Here we seek to test the option of increased component sdAbs in these heteromultimers by testing different sdAb heterohexamers in which each of the six camelid sdAb components (VHHs) can neutralize one of three different Botulinum neurotoxin (BoNT) serotypes, A, B or E. Each heterohexamer bound all three targeted BoNT serotypes and protected mice from at least 100 MIPLD50 of each serotype. To test the potential of mRNA therapeutics encoding long sdAb heteromultimers, one heterohexamer was encoded as replicating RNA (repRNA), formulated with a cationic nanocarrier, and delivered to mice via intramuscular injection. Heterohexamer antitoxin serum expression levels were easily detected by 8 h post-treatment, peaked at 5-10 nM around two days, and persisted for more than three days. Mice treated with the formulated repRNA one day post-treatment survived challenge with 100 MIPLD50 of each toxin serotype, demonstrating the function of all six component VHHs. Use of long sdAb multimers, administered as proteins or repRNA, offer the potential for substantially improved versatility in the development of antibody-based therapeutics.
Subject(s)
Antitoxins , Botulinum Toxins , Single-Domain Antibodies , Animals , Botulinum Toxins/genetics , Mice , RNA , Serogroup , Single-Domain Antibodies/geneticsABSTRACT
Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.
Subject(s)
Botulinum Toxins/antagonists & inhibitors , Peptide Hydrolases/chemistry , Single-Domain Antibodies , Animals , Botulinum Toxins/chemistry , Protease Inhibitors/pharmacology , Protein Domains/drug effects , Single-Domain Antibodies/pharmacology , Structure-Activity RelationshipABSTRACT
Typhoid toxin is secreted by the typhoid fever-causing bacterial pathogen Salmonella enterica serovar Typhi and has tropism for immune cells and brain endothelial cells. Here, we generated a camelid single-domain antibody (VHH) library from typhoid toxoid-immunized alpacas and identified 41 VHHs selected on the glycan receptor-binding PltB and nuclease CdtB. VHHs exhibiting potent in vitro neutralizing activities from each sequence-based family were epitope binned via competition enzyme-linked immunosorbent assays (ELISAs), leading to 6 distinct VHHs, 2 anti-PltBs (T2E7 and T2G9), and 4 anti-CdtB VHHs (T4C4, T4C12, T4E5, and T4E8), whose in vivo neutralizing activities and associated toxin-neutralizing mechanisms were investigated. We found that T2E7, T2G9, and T4E5 effectively neutralized typhoid toxin in vivo, as demonstrated by 100% survival of mice administered a lethal dose of typhoid toxin and with little to no typhoid toxin-mediated upper motor function defect. Cumulatively, these results highlight the potential of the compact antibodies to neutralize typhoid toxin by targeting the glycan-binding and/or nuclease subunits.
Subject(s)
Camelids, New World , Single-Domain Antibodies , Typhoid Fever , Animals , Endothelial Cells , Mice , Polysaccharides , Salmonella typhi , Typhoid Fever/microbiologyABSTRACT
For the developing field of gene therapy the successful address of the basic requirement effective gene delivery has remained a critical barrier. In this regard, the "Holy Grail" vector envisioned by the field's pioneers embodied the ability to achieve efficient and specific in vivo gene delivery. Functional linkage of antibody selectivity with viral vector efficiency represented a logical strategy but has been elusive. Here we have addressed this key issue by developing the technical means to pair antibody-based targeting with adenoviral-mediated gene transfer. Our novel method allows efficient and specific gene delivery. Importantly, our studies validated the achievement of this key vectorology mandate in the context of in vivo gene delivery. Vectors capable of effective in vivo delivery embody the potential to dramatically expand the range of successful gene therapy cures.
Subject(s)
Adenoviridae , Single-Domain Antibodies , Adenoviridae/genetics , Gene Transfer Techniques , Genetic Engineering , Genetic Therapy , Genetic Vectors , Single-Domain Antibodies/geneticsABSTRACT
Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.
Subject(s)
Cryptosporidiosis , Cryptosporidium parvum , Protozoan Proteins/genetics , Animals , Cryptosporidium parvum/genetics , Membrane Proteins/genetics , SporozoitesABSTRACT
Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism. Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin-based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol. A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons. Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice. These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.
Subject(s)
Botulism , Immunotoxins , Single-Domain Antibodies , Animals , Botulism/drug therapy , Disease Models, Animal , Mice , Synaptic TransmissionABSTRACT
Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.
Subject(s)
Botulinum Toxins, Type A , Botulism , Single-Domain Antibodies , Animals , Botulism/drug therapy , Guinea Pigs , Mice , Models, Animal , NeurotoxinsABSTRACT
Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.
Subject(s)
Antibodies, Neutralizing/pharmacology , Botulinum Toxins/antagonists & inhibitors , Botulism/prevention & control , Camelids, New World/immunology , Neurons/drug effects , Peptide Hydrolases , Protease Inhibitors/pharmacology , Single-Domain Antibodies/pharmacology , Animals , Antibodies, Neutralizing/immunology , Antibody Specificity , Binding Sites, Antibody , Botulinum Toxins/administration & dosage , Botulinum Toxins/immunology , Botulism/immunology , Botulism/microbiology , Cells, Cultured , Disease Models, Animal , Immunization , Male , Mice , Neurons/metabolism , Neurons/pathology , Peptide Hydrolases/administration & dosage , Peptide Hydrolases/immunology , Protease Inhibitors/immunology , Rats , Single-Domain Antibodies/immunologyABSTRACT
Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.
Subject(s)
Antibodies, Neutralizing/pharmacology , Botulinum Toxins/antagonists & inhibitors , Cell Membrane/drug effects , Membranes, Artificial , Single-Domain Antibodies/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibody Specificity , Biological Transport , Botulinum Toxins/genetics , Botulinum Toxins/immunology , Botulinum Toxins/metabolism , Cell Membrane/metabolism , Epitopes , Host-Pathogen Interactions , Mutation , Protein Conformation , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Structure-Activity RelationshipABSTRACT
Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.
Subject(s)
Antitoxins/chemistry , Botulinum Toxins/antagonists & inhibitors , Drug Design , Single-Domain Antibodies/chemistry , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Botulinum Toxins/chemistry , Cell Membrane/metabolism , Female , Hydrogen-Ion Concentration , Mice , Models, Molecular , Protein Domains , Protein Folding , Protein Multimerization , Receptors, Cell Surface/metabolismABSTRACT
We have recently developed a sensitive and specific urine-based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li-isd1), tryparedoxin1 (Li-txn1) and nuclear transport factor 2 (Li-ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li-isd1 and Li-ntf2. This new assay was specific and had analytical sensitivity of 15-45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li-txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.