Neutralization mechanism of human monoclonal antibodies against type B botulinum neurotoxin.

Botulism is a deadly neuroparalytic condition caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum and related species. Toxin-neutralizing antibodies are the most effective treatments for BoNT intoxication. We generated human monoclonal antibodies neutralizing type B botulinum neurotoxin (BoNT/B), designated M2 and M4. The combination of these antibodies exhibited a strong neutralizing effect against BoNT/B toxicity. In this study, we analyzed the mechanisms of action of these antibodies in vitro. M4 binds to the C-terminus of the heavy chain (the receptor-binding domain) and inhibits BoNT/B binding to neuronal PC12 cells. Although M2 recognized the light (L) chain (the metalloprotease domain), it did not inhibit substrate (VAMP2) cleavage in the cleavage assay. M2 increased the surface localization of BoNT/B in PC12 cells at a later time point, suggesting that M2 inhibits the translocation of the L chain from synaptic vesicles to the cytosol. These results indicate that M2 and M4 inhibit the different processes of BoNT/B individually and that multistep inhibition is important for the synergistic effect of the combination of monoclonal antibodies. Our findings may facilitate the development of effective therapeutic antibodies against BoNTs.

Novel platform for engineering stable and effective vaccines against botulinum neurotoxins A, B and E.

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most toxic protein known, capable of causing severe paralysis and posing a significant bioterrorism threat due to its extreme lethality even in minute quantities. Despite this, there are currently no FDA-approved vaccines for widespread public use. To address this urgent need, we have developed an innovative vaccine platform by fusing the neuronal binding domain of BoNT/E (Hc/E) with core-streptavidin (CS), resulting in a stable CS-Hc/E vaccine. Mice vaccinated with CS-Hc/E exhibited superior antibody titers compared to those receiving Hc/E alone. To develop a trivalent vaccine against BoNT/A, BoNT/B, and BoNT/E- key contributors to the vast majority of human botulism-we conjugated CS-Hc/E with a biotinylated atoxic chimeric protein incorporating neutralizing epitopes from BoNT/A and BoNT/B. This chimeric protein includes the binding domain of BoNT/A, along with the protease-inactive light chain and translocation domains of BoNT/B. The interaction between CS and biotin formed a stable tetrameric antigen, EBA. Vaccination with EBA in mice elicited robust antibody responses and provided complete protection against lethal doses of BoNT/A, BoNT/B, and BoNT/E. Our findings highlight EBA's potential as a stable and effective broad-spectrum vaccine against BoNT. Moreover, our technology offers a versatile platform for developing multivalent, stable vaccines targeting various biological threats by substituting the BoNT domain(s) with neutralizing epitopes from other life-threatening pathogens, thereby enhancing public health preparedness and biodefense strategies.

Immunogenicity of Botulinum Toxin A: Insights.

BACKGROUND: Botulinum toxin A (BoNT-A) is widely used in treating dystonia and spasticity to managing chronic migraine and cosmetic applications. However, its immunogenic potential presents challenges, such as the development of neutralizing antibodies that lead to diminished therapeutic efficacy over time, known as secondary nonresponse. OBJECTIVE: This review aims to bridge the knowledge gap regarding the immunogenic mechanisms of BoNT-A and to explore effective management strategies to mitigate these immune responses. MATERIALS AND METHODS: The authors conducted a systematic search in databases including PubMed, Embase, and Web of Science, using keywords related to BoNT-A's immunogenicity. The selection process refined 157 initial articles down to 23 relevant studies, which underwent analysis to investigate the underlying mechanisms of immunogenicity and the factors influencing it. RESULTS: The analysis revealed that both the neurotoxin component and the neurotoxin-associated proteins could elicit an immune response. However, only antibodies against the core toxin influence therapeutic outcomes. Various patient-specific factors such as genetic predispositions and prior immune experiences, along with treatment-related factors such as dosage and frequency, play crucial roles in shaping these responses. CONCLUSION: Understanding the specific immunogenic triggers and responses to BoNT-A is critical for optimizing treatment protocols and improving patient outcomes.

Mitigating Immunogenicity by Coordinating Botulinum Toxin Treatments Between Aesthetics and Therapeutics.

BACKGROUND: Aesthetic use of botulinum toxin (BoNT) has expanded greatly beyond conventional low-dose (20 U) treatments, leading to some patients receiving doses previously reserved for therapeutic uses. The resulting risks are compounded in patients who receive BoNT for both aesthetic and therapeutic indications. Implementing tools for risk management is a high priority to prevent reduced treatment duration and effectiveness. OBJECTIVE: To highlight the immunogenic risks of higher doses, with special attention to the compounding risks of resistance in patients with overlapping BoNT treatments from aesthetic and therapeutic indications. METHODS: Authors examined the literature on current practices to provide a side-by-side comparison of BoNT doses for aesthetic and therapeutic indications. RESULTS: Aesthetic BoNT doses used in combination treatments of multiple areas or single treatments of large muscle areas can meet or exceed those observed in therapeutic treatments. CONCLUSION: Physicians have a responsibility to incorporate risk management and open dialog into their BoNT treatment plans to maximize effectiveness and longevity of treatments.

Selection of Candidate Monoclonal Antibodies for Therapy of Botulinum Toxin Type A Intoxications.

Botulism is one of the most serious food intoxications, manifesting as prolonged paralytic conditions. This disease is usually the result of the consumption of poor quality canned or smoked foods, so the inhabitants of many countries of the world are exposed to the risk of this kind of poisoning every year. In view of the severity of poisonings caused by botulinum neurotoxins, monoclonal antibodies (mAbs) show great promise because of their targeting action, lack of allergic reactions and serum sickness. The use of a cocktail of mAbs increases the "functional specificity" of their mixture, allowing them to bind to the active domains of different toxin chains and block their action. In this work, we obtained 14 murine mAbs to the catalytic and receptor-binding domain of botulinum toxin type A. The Sp2/0-Ag14 murine myeloma cell line and splenocytes from immunized mice of the BALB/c line were used as fusion partners. We have shown that the selected cocktail of three antibodies neutralizes native toxin more effectively than antibodies separately-complete neutralization is achieved at a toxin dose of 3LD50 and partial neutralization at 5LD50. We presume that this cocktail may be promising as a prototype for the creation of a therapeutic drug capable of neutralizing the toxin in the blood of patients.

Design and evaluation of mRNA encoding recombinant neutralizing antibodies for botulinum neurotoxin type B intoxication prophylaxis.

Among all natural and synthetic toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botulinum in an anaerobic environment, are the most toxic polymer proteins. Currently, the most effective modalities for botulism prevention and treatment are vaccination and antitoxin use, respectively. However, these modalities are associated with long response time for active immunization, side effects, and donor limitations. As such, the development of more promising botulism prevention and treatment modalities is warranted. Here, we designed an mRNA encoding B9-hFc - a heavy-chain antibody fused to VHH and human Fc that can neutralize BoNT serotype B (BoNT/B) effectively - and assessed its expression in vitro and in vivo. The results confirmed that our mRNA demonstrates good expression in vitro and in vivo. Moreover, a single mRNA lipid nanoparticle injection effectively prevents BoNT/B intoxication in vivo, with effects comparable to those of protein antibodies. In conclusion, we explored and clarified whether mRNA drugs encoding neutralizing antibodies prevent BoNT/B intoxication. Our results provide an efficient strategy for further research on the prevention and treatment of intoxication by botulinum toxin.

Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B.

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Reducing the Impact of Headache and Allodynia Score in Chronic Migraine: An Exploratory Analysis from the Real-World Effectiveness of Anti-CGRP Monoclonal Antibodies Compared to Onabotulinum Toxin A (RAMO) Study.

BACKGROUND: Chronic migraine (CM) is a disabling and hard-to-treat condition, associated with high disability and high cost. Among the preventive treatments, botulinum toxin A (BoNT-a) and monoclonal antibodies against the calcitonin gene-related protein (anti-CGRP mAbs) are the only disease-specific ones. The assessment of the disease burden is complex, and among others, tools such as the allodynia symptoms checklist (ASC-12) and headache impact test (HIT-6) are very useful. This exploratory study analysed the impact of these two therapies on migraine burden. METHODS: The RAMO study was a multicentre, observational, retrospective investigation conducted in two headache centres: the Fondazione IRCCS Istituto Neurologico Carlo Besta (Milan) and the Fondazione Policlinico Campus Bio-Medico (Rome). This study involved patients with chronic migraine treated with mAbs or BoNT-A. We conducted a subgroup exploratory analysis on HIT-6 and ASC-12 scores in the two groups. The Wilcoxon rank-sum test, Fisher's exact test, and ANOVA were performed. RESULTS: Of 126 patients, 36 on mAbs and 90 on BoNT-A had at least one available follow-up. mAbs resulted in a mean reduction of -11.1 and -11.4 points, respectively, in the HIT-6 at 6 and 12 months, while BoNT-A was reduced -3.2 and -3.6 points, respectively; the mAbs arm resulted in mean reductions in ASC-12 at 6 and 12 months of follow-up of -5.2 and -6.0 points, respectively, while BoNT-A showed lesser mean changes of -0.5 and -0.9 points, respectively. The adjusted analysis confirmed our results. CONCLUSIONS: In this exploratory analysis, anti-CGRP mAbs showed superior effectiveness for HIT-6 and ASC12 compared to BoNT-A. Reductions in terms of month headache days (MHD), migraine disability assessment test (MIDAS), and migraine acute medications (MAM) were clinically relevant for both treatments.

Sub-acute hypersensitive reaction to botulinum toxin type A following Covid-19 vaccination: Case report and literature review.

RATIONALE: Botulinum toxin type A (BTA) is one of the most widely used injectable agents in cosmetic surgery. Corona virus disease 2019 (Covid-19) infection and vaccination, which can induce specific and nonspecific activation of the immune system, has been reported to induce delayed inflammatory reactions to previously injected hyaluronic acid fillers. However, there are no reports about the interaction between BTA and Covid-19. We aimed to report 2 sub-acute cases of allergic reactions to BTA in facial cosmesis following the Covid-19 vaccination. PATIENT CONCERN: A 35-year-old and a 34-year-old female who has several previous BTA injections without any adverse effects experienced facial swelling, flu-like symptoms after BTA treatment following the Covid-19 vaccination. DIAGNOSE: According to the typical clinical manifestation, a hypersensitive reaction to BTA was considered. INTERVENTION: Corticosteroids and antihistamine were administered empirically. OUTCOMES: The flu-like symptoms recovered over the next day, but the facial swelling gradually faded within 1 to 2 weeks. LESSONS: A literature review was also conducted to summarize the hypersensitive actions to cosmesis related to Covid-19. We recommend BTA injection be administered at least 2 to 3 months after Covid-19 vaccination.

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.

Recombinant L-HN Fusion Antigen Derived from the L and HN Domains of Botulinum Neurotoxin B Stimulates a Protective Antibody Response Against Active Neurotoxin.

Botulinum neurotoxin (BoNT) is a neurotoxin produced by Clostridium botulinum in an anaerobic environment. BoNT is the most toxic protein among bacteria, animals, plants, and chemical substances reported to date. BoNTs are 150 kDa proteins composed of three major functional domains: catalytic (L domain, 50 kDa), translocation (HN domain, 50 kDa), and receptor-binding (Hc domain, 50 kDa) domains. Most studies have focused on the use of the Hc domain as an antigen because it is capable of generating robust protective immunity and contains some functional neutralizing epitopes. In the present study, we produced and characterized a recombinant L-HN fusion fragment of the parent BoNT/B (BL-HN) composed of L and HN domains with a deletion in the Hc domain (BHc). When the BL-HN protein was expressed in E. coli, it retained its stable structure and antigenicity. As a vaccine antigen, the recombinant BL-HN protein was found to induce sufficient protection against native BoNT/B in a mouse model. The BL-HN subunit vaccine could also induce a strong humoral immune response and generate sufficient neutralizing antibodies in immunized mice. Therefore, BL-HN may retain the native neurotoxin structure and critical epitopes responsible for inducing serum neutralizing antibodies. Studies of the dose-dependent immunoprotective effects further confirmed that the BL-HN antigen could provide potent protective immunity. This finding suggests that BL-HN can play an important role in immune protection against BoNT/B. Therefore, the BL-HN fusion fragment provides an excellent platform for the design of recombinant botulinum vaccines and neutralizing antibodies.

Production and characterization of a neutralizing antibody against botulinum neurotoxin A.

As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease 'botulism' in human and animals. Their potential use as biological weapon led to their classification in category 'A' biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni-NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

Can clinical and urodynamic parameters predict the occurrence of neutralizing antibodies in therapy failure of intradetrusor onabotulinumtoxin A injections in patients with spinal cord injury?

BACKGROUND: The aim of the study was to clarify whether clinical and/or urodynamic parameters could be used to infer the probability of neutralizing antibody (NAb) formation as a possible cause of therapy failure (non-response, NR) in patients with neurogenic detrusor overactivity (NDO) due to acquired spinal cord injury/disease (SCI/D) treated with intradetrusor botulinum neurotoxin A (BoNT-A) injections. METHODS: A retrospective chart review was performed of all patients with SCI/D who underwent both intradetrusor onabotulinumtoxin A injections and the determination of neutralizing antibodies against BoNT-A between January 1, 2002, and December 31, 2018. NR was defined as urodynamically confirmed persistent or reappearing NDO. RESULTS: A total of 2700 BoNT-A injections in 414 patients were ascertained. In 69 patients with primary NR after the first BoNT-A injection (n = 6) or with secondary NR after more than one BoNT-A injection (n = 63), an antibody analysis was performed. Antibody examination showed 36 (52.2%) negative, 5 (7.2%) borderline and 14 (each 20.3%) each of positive and highly positive values. Subgroup analysis indicated a correlation between NAb formation and the duration of BoNT-A therapy (p = 0.015), the mean number of BoNT-A injections (p = 0.011) and the time interval between BoNT-A applications (< 7 months, p = 0.022). Urodynamic data analysis indicate significant differences with cut-off values of MCC (< 225 ml, p = 0.038) and MDP (> 45 cmH2O, p = 0.040). However, in the regression analysis models, the predictive value for the occurrence of NAb was too low (MCC: ROC AUC 0.62, MDP: ROC AUC 0.52) to distinguish with sufficient certainty between NAb-positive and NAb-negative NR patients. CONCLUSIONS: Despite significant correlations, clinical and urodynamic parameters are only partially suitable for predicting antibody formation against BoNT-A.

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B.

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that mediate their effects by binding to neuronal receptors and block the neutralizing ability of therapeutic antibodies. Vaccination is currently the most effective strategy to prevent botulism. In this study, a series of recombinant functional domain antigens of BoNT/A were prepared and identified, and their immunoprotective efficacies were explored and compared. Our results showed that all antigens produced strong humoral immune responses, although their protective effects against the toxin were different. Only the Hc and HN-L antigens produced strong protective effects and afforded complete immunoprotection. In addition, the combined vaccine groups showed that there was no synergistic effect on immune responses after antigen combination, suggesting that the integrity of the toxin antigen or domain is crucial to the immune effects. Studies of the dose-dependent immunoprotective effects further confirmed that the Hc domain antigen afforded more effective protective potency than the HN-L antigen, equivalent to the immune effect of the full-length toxin (Hc + HN-L combination group). Overall, our results demonstrated that the Hc domain elicited a strong protective immune response and also provided basic data and theoretical support for the development of Hc-based BoNT/A subunit vaccine. Therefore, the receptor binding domain Hc is implicated as a promising target antigen of the BoNT/A recombinant subunit vaccine as an alternative to the toxoid vaccine.

Effective long-term treatment with incobotulinumtoxin (Xeomin®) without neutralizing antibody induction: a monocentric, cross-sectional study.

BACKGROUND: Among the spectrum of licensed botulinum neurotoxin preparations incobotulinumtoxin (incoBoNT/A; Xeomin®) is the only one which does not contain complex proteins. Therefore, incoBoNT/A has been suggested to have a low antigenicity, but precise estimations on incidence and prevalence of neutralizing antibody formation during long-term treatment are outstanding so far. METHODS: For the present cross-sectional study, 59 patients having exclusively been treated with incoBoNT/A (mono group) and 32 patients having been treated with other BoNT/A preparations less than nine times and who were then switched to at least 14 sessions of incoBoNT/A treatment (switch group) were recruited from one botulinum toxin outpatient clinic. Side effects and doses were extracted from the charts, and the efficacy of treatment was assessed by the patients using a visual analogue scale (0-100). The prevalence of neutralizing antibodies was tested by means of the mouse hemi-diaphragm assay (MHDA). FINDINGS: None of the patients in the mono and only two in the switch group had a positive MHDA-test. Across all indications and patients, mean improvement exceeded 67%. Improvement did not depend on age at onset, sex, change of dose or duration of treatment, but on disease entity. In patients with cervical dystonia, improvement was about the same in the mono and switch subgroup, but the last dose was different. CONCLUSIONS: The present study confirms the low antigenicity of incoBoNT/A, which has immediate consequences for patient management, and the use of higher doses and shorter durations of reinjection intervals in botulinum toxin therapy.

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice.

The bacterium Clostridium botulinum is the causative agent of botulism-a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.

Immunogenicity Associated with Botulinum Toxin Treatment.

Botulinum toxin (BoNT) has been used for the treatment of a variety of neurologic, medical and cosmetic conditions. Two serotypes, type A (BoNT-A) and type B (BoNT-B), are currently in clinical use. While considered safe and effective, their use has been rarely complicated by the development of antibodies that reduce or negate their therapeutic effect. The presence of antibodies has been attributed to shorter dosing intervals (and booster injections), higher doses per injection cycle, and higher amounts of antigenic protein. Other factors contributing to the immunogenicity of BoNT include properties of each serotype, such as formulation, manufacturing, and storage of the toxin. Some newer formulations with purified core neurotoxin devoid of accessory proteins may have lower overall immunogenicity. Several assays are available for the detection of antibodies, including both structural assays such as ELISA and mouse-based bioassays, but there is no consistent correlation between these antibodies and clinical response. Prevention and treatment of antibody-associated non-responsiveness is challenging and primarily involves the use of less immunogenic formulations of BoNT, waiting for the spontaneous disappearance of the neutralizing antibody, and switching to an immunologically alternate type of BoNT.

Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 µL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 µL of equine serum and 100 µg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.