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.

Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most toxic known protein and the causative agent of human botulism. BoNTs have similar structures and functions, comprising three functional domains: catalytic domain (L), translocation domain (HN), and receptor-binding domain (Hc). In the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. The EL-HN fragment (L and HN domains of BoNT/E) retained the enzymatic activity without in vivo neurotoxicity. Extensive investigations showed EL-HN functional fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Further experiments demonstrated the EL-HN provided a superior protective effect compared with the EHc or EHc and EL-HN combination. Thus, the EL-HN played an important role in immune protection against BoNT/E and could provide an excellent platform for the design of botulinum vaccines and neutralizing antibodies. The EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism.

OBJECTIVES: To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. RESULTS: In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. CONCLUSIONS: Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.

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.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

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.

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.

Recombinant vaccine against botulism in buffaloes: Evaluation of the humoral immune response over 12 months.

Botulism is a neuroparalytic intoxication, usually fatal, caused by the botulinum toxins (BoNTs). Vaccination is the best-known strategy to prevent this disease in ruminants. Serotypes C and D and their variants CD and DC are the main types responsible for botulism in bovine and buffaloes in Brazil and cattle in Japan and Europe. Brazil has a herd of approximately 1.39 million buffaloes and is the largest producer in the Western world. This study aimed to assess the humoral immune response of buffaloes during the 12-month period after vaccination against BoNT serotypes C and D with a recombinant vaccine in three different concentrations (100, 200, and 400 µg) of non-purified recombinant proteins (Vrec) and also with a bivalent commercial toxoid (Vcom). Vrec400 was the best vaccine among those tested because it induced higher levels of antibodies and maintained higher levels of antibodies for the longest time, while Vrec200 could be considered the most cost-effective vaccine for large-scale production. None of the vaccines were able to promote continuous immunological protection within the timeframe proposed by the current Brazilian vaccination protocol. Further studies should focus on vaccine adjustments to ensure continued humoral protection against botulism.

An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E.

Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.

Safety and immunogenicity of investigational recombinant botulinum vaccine, rBV A/B, in volunteers with pre-existing botulinum toxoid immunity.

OBJECTIVES: We undertook an open-label, uncontrolled study of investigational recombinant botulinum vaccine for botulinum neurotoxin (BoNT) serotypes A and B (rBV A/B) to assess its safety and immunogenicity in healthy volunteers who had been previously immunized with investigational pentavalent botulinum toxoid. Study participants who wished to do so could donate their hyperimmune plasma for production of Human Botulism Immune Globulin Intravenous (BIG-IV, BabyBIG®). STUDY DESIGN: A single 0.5 ml (mL), 40-microgram intramuscular injection of rBV A/B was administered to study participants. Post-vaccination sera collected at approximately 2-week intervals were evaluated for anti-BoNT/A and anti-BoNT/B neutralizing antibody concentrations (NAC). Local and systemic treatment-emergent adverse events (TEAEs) were identified by clinical and laboratory monitoring for 12 weeks post-vaccination with a final telephone follow-up for additional safety assessment at 6 months. The primary endpoint for immunogenicity was a ≥4-fold rise in NAC in ≥50% of participants by Week 4 post-vaccination. RESULTS: All 45 enrolled participants completed the study. Forty-two of 45 participants (93.3%) experienced at least one TEAE. Overall, 138 of 218 (63.3%) reported TEAEs were treatment-related, the majority of which were mild injection-site reactions. No serious or unexpected adverse events occurred. The study achieved its primary immunogenicity endpoint with 37/45 (82.2%) participants and 39/45 (86.7%) participants having a ≥4-fold rise in NAC to anti-BoNT/A and to anti-BoNT/B, respectively, by Week 4 post-vaccination. CONCLUSION: A single 0.5 mL dose of rBV A/B was safe, well-tolerated and immunogenic in participants previously immunized with pentavalent botulinum toxoid. The tolerability and immunogenicity characteristics of rBV A/B vaccination of individuals with existing BoNT immunity support its potential future use to provide occupational protection to botulism laboratory workers. Almost all study participants donated hyperimmune plasma for production of BIG-IV. ClinicalTrials.gov registration number: NCT01701999.

Identification of Cross Reactive Antigens of C. botulinum Types A, B, E & F by Immunoproteomic Approach.

Diseases triggered by microorganisms can be controlled by vaccines, which need neutralizing antigens. Hence, it is very crucial to identify extremely efficient immunogens for immune prevention. Botulism, a fatal neuroparalytic disease, is caused by botulinum neurotoxins produced by the anaerobic, Gram-positive spore-forming bacteria, Clostridium botulinum. Food-borne botulism and iatrogenic botulism are caused by botulinum toxin. Wound botulism, infant botulism, and adult intestinal botulism are caused by primarily C. botulinum followed by secondary intoxication. To identify protective antigens, whole cell proteome of C. botulinum type B was separated by two-dimensional gel electrophoresis. 2-D gel of whole cell proteins was probed with hyper immune sera of whole cell proteins of C. botulinum types A, E, and F. Six cross immunoreactive proteins were identified. These immunoreactive proteins will be further tested for developing vaccines and serodiagnostic markers against botulism.

Enhancing toxin-based vaccines against botulism.

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HCN) and a receptor binding domain (HCC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHCN/A1 and HCC/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >106-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1W. M-BoNT/A1W vaccination protected against challenge by 106 LD50 Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1W surviving BoNT challenge had dominant antibody responses to the LCHCN domain, but varied antibody responses to HCC. Sera from mice vaccinated with M-BoNT/A1W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.

Nanogel-based nasal vaccines for infectious and lifestyle-related diseases.

Because the mucosa is the major entry route for most pathogens, the development of mucosal vaccines is a rational approach for protecting against these undesired agents. Mucosal administration of vaccine antigen is useful for non-infectious chronic diseases as well, because of its advantages over injection routes, including comparable efficacy in the induction of systemic immune responses, less pain, and no risk of adverse events at the injection site. However, because it is difficult to effectively induce and regulate antigen-specific mucosal and systemic immune responses when antigen alone is mucosally administered, an appropriate form of mucosal delivery vehicle must be used. Antigen delivery systems involving nanogels, which act as artificial chaperones and mucosal adhesives, are a promising approach to overcoming this problem. Here, we introduce current perspectives regarding the development of nanogel-based nasal vaccines for both infectious and lifestyle-related diseases.

Therapeutic Plasma Exchange in a rare case myasthenic crisis after Botox injection.

BACKGROUND AND AIMS: Botulinum toxin (Botox) injections are used as a cosmetic treatment to decrease wrinkles in face and chin. Being a neurotoxic agent it minimizes muscle activity, while side effects are usually rare. This article subsequently presents one case of these rare effects. CASE: A 30-year-old woman presenting with ptosis, diplopia, dysarthria, dysphagia and muscle weakness was admitted to our hospital. She had no history of disease. For cosmetic reasons, she had three Botox injections during the preceding months. On physical examination, muscle weakness 4/5 (cervical extensor, ocular and pharynx) was detected and a diagnosis of myasthenia gravis was made. Protective artificial ventilation was necessary. As a consequence, eight sessions of 2.5 L volume Therapeutic Plasma Exchange (TPE) were applied using normal saline/albumin as substitute. Due to TPE, her muscle force and clinical condition improved. Artificial ventilation could be stopped. CONCLUSIONS: Clinical symptoms of myasthenia gravis and systemic Botox effects are very similar. This should be taken into consideration during medical history taking. The injection of high doses of Botox (more than 200 units in every injection) or boostering within less than one month is dangerous. (Botox BCC2024). Systemic side effects can be treated using TPE to lower the circulating dose of Botox.

Therapeutic efficacy of equine botulism antitoxin in Rhesus macaques.

BACKGROUND: There are currently no licensed vaccines available for prevention of botulism in humans. The vaccination is not desirable due to expanding therapeutic indications of botulinum toxins. The only available specific treatment for botulism is antitoxin to remove circulating toxin, thus, preventing further neuronal damage. BAT® (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)) has been developed and its therapeutic efficacy evaluated against botulinum neurotoxin serotype A (BoNT/A) in Rhesus macaques. METHODS AND FINDINGS: In a post-exposure prophylaxis (PEP) study, animals were exposed to 4x LD50/kg of BoNT/A and administered intravenously with either BAT (1x or 0.1x scaled human dose), or placebo at 4 hours post-exposure. The animals were monitored for 14 days. For the therapeutic intervention studies, animals were exposed to a 1.7x LD50/kg of BoNT/A and treated intravenously with either placebo or BAT at a 1x scaled human dose at the onset of clinical signs. Animals were monitored on an hourly basis for 14 or 21 days. In the PEP study, all animals tolerated equine based antitoxin without any adverse clinical signs. A 100% survival was observed in groups treated with the BAT compared to 0% survival in those treated with the placebo (p<0.001, Fisher's exact test). BAT antitoxin prevented the development of signs of neurotoxicity of botulinum toxin. In a therapeutic study, treatment with the BAT at scaled 1x human dose after the onset of clinical signs significantly enhanced survival compared to the placebo (46.6% vs. 0%, p<0.0001, Fisher's exact test). Additionally, treatment with the BAT delayed the progression of signs (muscular weakness, respiratory distress, oral/nasal discharge) of toxin intoxication and reduced the severity of the disease. CONCLUSIONS: A single dose of BAT, when administered to symptomatic monkeys, resulted in a statistically significant survival benefit compared to the placebo. Additionally, BAT completely protected monkeys from the clinical signs of intoxication and subsequent death when administered as PEP treatment. These data in part supported the licensure of BAT under the Animal Rule in the United States by the Food and Drug Administration.

Enhanced effects of DNA vaccine against botulinum neurotoxin serotype A by targeting antigen to dendritic cells.

As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.

Immunoproteomics Approach for Screening of Vaccine Candidates against Intestinal Botulism.

Intestinal botulism is an infectious form of botulism in which disease results from ingesting spores, which is followed by spore germination and intraluminal production of botulinum neurotoxins over an extended period. Botulinum neurotoxin is produced by endospore forming bacteria called C. botulinum. Immunoproteomic study was used to screen the cross reactive immunogenic proteins of Clostridium botulinum type B using C. botulinum type B live spore antiserum. The whole cell proteins were separated by two dimensional gel electrophoresis and transferred to polyvinylidene difluoride membranes. Further, the Western blotting was performed with mouse pups immune serum against C. botulinum type B live spores. Eight predominant cross immunoreactive proteins were identified by mass spectrometry. These immunogenic proteins might be used to develop novel subunit vaccine candidates against the intestinal botulism.

Clinical Serum Therapy: Benefits, Cautions, and Potential Applications.

Blood serum from immunized humans or animals (e.g., horses) contains relevant antibodies and has been used as serum therapy to treat many diseases or envenomation events. The effectiveness of blood serum was initially discovered in 1890 when Kitasato and von Behring observed the effectiveness of this type of therapy against diphtheria and tetanus. Serum therapies played an important role in the advancement of modern medicine prior to the development of penicillin and steroids. At present, several types of serum therapy remain in clinical use. However, some physicians have a limited understanding of the nature and the benefits of serum therapy and the factors that require particular attention. In this review, we set out to clarify the benefits, cautions, and potential applications of serum therapy in the context of conditions such as gas gangrene, diphtheria, botulism, and tetanus and bites from three snake species (mamushi, habu, and yamakagashi) and the redback spider. It is hoped that this review will help clinicians to learn about clinical serum therapies and become familiar with their applications.

In-Vivo Neutralization of Botulinum Neurotoxin Serotype E Using Rabbit Polyclonal Antibody Developed against BoNT/E Light Chain.

BACKGROUND: Clostridium botulinum is an obligate anaerobic, Gram positive bacterium that secretes extremely toxic substances known as botulinum neurotoxins (BoNTs) that cause serious paralytic illness called botulism. Based upon the serological properties, these neurotoxin have been classified into seven serotypes designated from A to G. Due to extreme toxicity of BoNTs, these neurotoxins have been designated as category A biowarfare agents. There is no commercial neutralizing antibody available for the treatment of botulism. Hence there is an urgent need to develop therapeutic intervention for prevention and cure of botulism within short period. BoNT antiserum injection is still the effective treatment. METHOD: In the present study, the recombinant light chain of BoNT/E was successfully purified in soluble form. The purified rBoNT/E LC was used for the generation of polyclonal antibody in rabbit. In order to find out the neutralizing capacity of generated antisera, rabbit antiserum was incubated with 20 LD50 of botulinum neurotoxin type E for 1 hour at 37°C and then injected intraperitoneally (IP) into mice. Further in another set of experiments antiserum was administered in different ways that included administration of - antiserum and BoNT/E toxin simultaneously without preincubation, one after another at the same and different time points for its therapeutic ability. To find out cross neutralization capacity, rBoNT/E LC antiserum was pre-incubated with 5 LD50 of BoNT/A, BoNT/B, BoNT/F and then injected (IP) into mice. In all the cases mice were observed continuously for 96 hours. RESULT: The results clearly indicate that developed polyclonal rabbit antiserum showed serotype specific neutralization of BoNT/E toxin only but not of BoNT/A, BoNT/B and BoNT/F. CONCLUSION: The developed antibodies will be used for preventive and therapeutic intervention of type 'E' botulism.