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. 2023 Apr 14;107(10):3205–3216. doi: 10.1007/s00253-023-12515-2

Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs

Dan-Yang Shi 1,2,#, Jian-Sheng Lu 1,#, Yun-Yun Mao 1,#, Fu-Jia Liu 1, Rong Wang 1, Peng Du 1, Shuo Yu 1, Yun-Zhou Yu 1,✉, Zhi-Xin Yang 1,✉
PMCID: PMC10102682  PMID: 37058230

Abstract

Abstract

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab′)2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins.

Key points

• Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made.

• Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain.

• A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-023-12515-2.

Keywords: Botulinum neurotoxin, Botulism, Receptor-binding domain, Tetravalent botulism antitoxin, Neutralizing antibody, Immunotherapy

Introduction

Botulism is a rare but serious illness caused by a neurotoxin produced by Clostridium botulinum (Sobel 2005; Hill et al. 2007; Rao et al. 2021). There are seven serotypes of botulinum neurotoxins (BoNTs): human botulism is caused by serotypes A, B, E, and F, while bird, horse, and cattle botulism are caused by serotypes C, D, and G, respectively. Recent research has identified new BoNT serotypes (such as BoNT/HA and BoNT/X) and BoNT-like proteins (Tehran and Pirazzini 2018), confirming BoNT variability and diversity.

BoNTs share a similar structure and function. Initially, they are produced as 150-kDa proteins, followed by cleavage into the 50-kDa light chain and the 100-kDa heavy chain, which are connected using disulfide bonds. BoNTs comprise three functional domains: the 50-kDa N-terminal catalytic domain (light chain, L), the 50-kDa internal heavy chain translocation domain (HN), and the 50-kDa C terminal heavy chain receptor-binding domain (Hc) (Pirazzini et al. 2017; Rossetto et al. 2019). Via circulation through the blood, the Hc binds to receptors on nerve ending membranes (Shukla and Sharma 2005), thereby triggering BoNT transfer into the neuronal cytoplasm via Hc-mediated translocation. In the cytoplasm, the light chain breaks down the proteins that are vital for neurotransmitter release, thus blocking the extracellular release of neurotransmitters into the neuromuscular junction, causing dyspnea, muscle paralysis, and even death (Dong and Stenmark 2021; Smith 2009).

Botulism has four naturally occurring syndromes: foodborne botulism, wound botulism, infant botulism, and adult intestinal colonization botulism. In addition, inhalational botulism could result from aerosolization of toxin, and iatrogenic botulism can result from high concentration cosmetic or therapeutic injections of toxin (Dembek et al. 2007; Rao et al. 2021; Sobel 2005). Botulism cases and outbreaks are public health problems that require vigorous attention. The measures of therapy are meticulous intensive care (including mechanical ventilation) and timely treatment with antitoxin. Botulinum antitoxin (BAT) is the only specific therapy for botulism. Antitoxins can bind the circulating BoNTs and halt illness progression, thereby blocking the toxic effects. Therefore, monovalent or multivalent antitoxins against BoNTs have been developed in different countries (Lonati et al. 2020; O’Horo et al. 2017). A heptavalent botulism antitoxin (H-BAT) derived from horse plasma was approved by the FDA in 2013 and is the only BAT that can treat all seven kinds of BoNTs ( Parrera et al. 2021). Currently, H-BAT is the only approved drug to treat botulism poisoning in adults or children in USA, the trivalent botulinum antitoxin (BoNT/A, B, E) used in Europe, and the monovalent botulinum antitoxin used in China (Parrera et al. 2021; Richardson et al. 2020; Yu et al. 2017). BabyBIG (botulism immunoglobulin), consisting of human-derived botulism antitoxin antibodies, was also approved, but only for the treatment of infant botulism from serotypes A and B (Long 2018), because human antiserum cannot meet the demand for large-scale preparation. BabyBIG is administered as a one-time 50-mg intravenous dose, and each dose is formulated to contain at least 15 IU of neutralizing antibodies against BoNT/A and 4 IU of neutralizing antibodies against BoNT/B. The neutralization potency of BabyBIG is only 1 ~ 4% of the antitoxins against BoNT/A and B (Rosow and Strober 2015; Shearer et al. 2010).

Genetically engineered monoclonal antibodies are also in the development stage (Raja et al. 2022; Rasetti-Escargueil et al. 2017; Tomic et al. 2021). However, technologically, equine-derived BAT is the most mature BAT available to date (Kim et al. 2019; Rasseti-Escargueil and Popoff 2019; Richardson et al. 2020). The H-BAT and other traditionary antitoxins were derived from the plasma of horses immunized with inactivated toxins or toxoid, which is harmful to the animals and limits the potency of the antitoxins. Previously, we developed botulinum vaccines for BoNT serotypes A, B, E, and F using recombinant BoNT Hc domains as antigens (Shi et al. 2022). These recombinant antigens proved to be high potent in eliciting strong immune responses in mice and horses (Shi et al. 2020; Yu et al. 2010a, 2010c), and the serum antibodies had high neutralizing potency against BoNTs.

Herein, we developed and characterized a novel equine tetravalent botulism antitoxin (T-BAT) based on the receptor-binding domains (RBDs) of BoNTs. These highly potent purified recombinant antigens were formulated with Freund’s adjuvant and injected to horses. Horse serum was collected, and the antibody titers were tested. A high-potency monovalent antitoxin was developed and tested in vitro and in vivo. Then, T-BAT was produced on a pilot scale by mixing the four monovalent antitoxins at suitable proportions. The novel T-BAT was then characterized and assayed using previously published methods (Shi et al. 2020; Yu et al. 2010a, 2010c), and its potency was also assessed in preclinical experiments.

Material and methods

Animals

Female Kunming (KM) mice weighing 15–18 g were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and raised in a pathogen free environment. We bought 40 healthy horses (weighing 300–350 kg; aged 4–6 years) from Chifeng Bo En Pharmaceutical Co., Ltd. (Chifeng, China), which were reared under standard conditions. The Animal Care and Use Committee (Beijing Institute of Biotechnology) approved all the animal experiments, which were carried out following the institute’s ethical guidelines and the National Institutes of Health guide for the care and use of Laboratory animals (IACUC-DWZX-2019–017).

Antigen preparation and characterization

The non-tagged recombinant BoNT Hc domain antigens were prepared as described previously (Shi et al. 2022). Briefly, E.coli was used to express BoNT/A, BoNT/E, and BoNT/F antigens (AHc, EHc, FHc); the yeast was used to express the BoNT/B antigen (BHc). The Pilot Production Base at our institute purified these antigens using sequential chromatography (pilot-scale batch of 30 L). The pilot-scale batches of Hc antigens were examined, and the batches of qualified antigens were stored at − 80 °C for further use.

Horse immunization and anti-RBD horse sera collection

Ten horses, in which no anti-RBD antibodies were detected, were immunized with each recombinant antigen. First, subcutaneous immunization was provided using 2 mg of the antigen in phosphate-buffered saline (PBS) emulsified using an equal volume of complete Freund’s adjuvant (CFA, Sigma, St. Louis, MO, USA). Subsequent immunizations used 2, 3, 3, and 4 mg of the antigen emulsified using the same volume of incomplete Freund’s adjuvant (IFA, Sigma) at 2–3-week intervals. The preparation was injected near the horses’ submandibular lymph nodes and inguinal lymph nodes to the rear of their bodies, with less than 2 ml being injected into each part. At 7 days post-immunization, serum samples were taken, and the antibody titers and neutralization titers were determined to assess the effect of immunization of the horses. According to the results of neutralization test, the qualified horses were bled to collect a large amount of high neutralization immune sera into sterile pyrogen free glass containers preloaded with anticoagulant glucose citrate (ACD). After 2 weeks of rest, the horses were immunized continuously with 5 ~ 6 mg of each antigen to produce more immunized sera to prepare the antitoxin until the twelfth immunization during 6 months.

Antibody titer and BoNT neutralization assays of horse sera

Enzyme-linked immunosorbent assays (ELISAs) were used to determine the anti-RBD antibody titer of the hyperimmune sera, according to a previously published method (Shi et al. 2022). The Pharmacopoeia of the People’s Republic of China (PPRC, Appendix XII H: the potency assay of botulinum antitoxin) was used to determine the neutralizing potency. In short, a gradient dilution of the toxin was mixed with 1 IU of a reference standard antitoxin for each BoNT (from the National Institutes of Food and Drug Control, Beijing, China) and injected intraperitoneally into mice (four mice per sub group) to determine the L + dose (defined as the minimum dose of toxin needed to kill the mice) of the BoNTs. Survival of the mice was recorded over 4 days. The determined L + dose of toxin was then mixed with diluted horse sera or antitoxin F(ab′)2 and injected into the mice to assess the neutralization effect. The neutralization potency of the antitoxin F(ab′)2 or horse sera was calculated in comparison with the BoNT reference standard antitoxins, and was reported as IU/ml following the WHO (World Health Organization) guidance regarding BoNT antitoxins.

Western blot assay

Western blot was used to detect the antigenicity of the recombinant proteins. Following separation using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the Hc, Hc-N, Hc-C, catalytic domain (L), translocation domain (HN), and L-HN domains of BoNTs (Li et al. 2021, 2022, 2023; Liu et al. 2020) were electrotransferred to a polyvinylidene fluoride (PVDF) membrane, followed by blocking at 37 °C for 1 h using 5% skim milk. Antibodies (1:500 diluted) from hyperimmune anti-BoNT toxoid (National Institutes of Food and Drug Control) or Hc horse sera were incubated with the membrane overnight at 4 °C. Following TBST (Tris-buffered saline with 0.1% Tween-20) washing, the membrane was incubated for 1 h at 37 °C with 1:2000 diluted goat anti-horse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Following further TBST washing, we added western blot solution (Thermo Fisher Scientific, Waltham, MA, USA) to the membrane, followed by exposure on a gel imager (Bio-Rad, Hercules, CA, USA).

Preparation and properties of monovalent BATs

According to previously published methods, we prepared total equine IgG and F(ab′)2 (Kim et al. 2019; Shi et al. 2020; Wang et al. 2005). Briefly, pyrogen-free distilled water was used to dilute the hyperimmune sera (1:2 v/v) and the pH was adjusted to 3.0 using 1 N HCl. Pepsin (6 U/ml) was used to digest the mixed equine IgG from 6 to 8 horses for 30 min at 37 °C. Then, the mixture was added with 15% (w/v) ammonium sulfate, adjusted to pH 5.4, and incubated for 30 min at 58 °C to precipitate unwanted proteins. Immediately following incubation, we cooled the mixture to 45 °C and added 0.8% perlite, stirred, and filtered it to obtain the filtrate. We adjusted the pH of the filtrate to 7.2, and ammonium sulfate was added to a final concentration of 20% (w/v), followed by incubation for 45 min. Next, we added 0.8% perlite, stirred, and press-filtered the mixture to obtain the sediment. We then dissolved the sediment in two volumes of pyrogen-free distilled water. Alum was then added to the mixture at a final proportion of 0.8%, and we adjusted the mixture to pH 7.8 ± 0.1. To adsorb the thermally denatured Fc fragments, the mixture was stirred for 60 min, followed by press-filtering. A centrifugal filtration device (Millipore, Billerica, MA, USA; molecular weight (MW) cut-off = 50 kDa) was used to further separate and concentrate the filtered F(ab′)2 fragment (Kittipongwarakarn et al. 2011; Morais and Massaldi 2005; Rial et al. 2006).

The obtained F(ab′)2 was balanced using PBS (pH 7.0), and the filtered through a 0.22 μm filter (Millipore). The final F(ab′)2 product was preserved at 4 °C before determination of its purity, concentration, potency, and specificity. Previously described methods were used to determine the neutralizing effect of the F(ab′)2 antitoxin (Wang et al. 2005; Yu et al. 2010a, b). A mouse in vivo protection model was used to assess the efficacy of the novel antitoxin.

Development and characterization of T-BATs

T-BATs were prepared by mixing four kinds of monovalent BATs under good manufacturing practice (GMP) conditions according to the previously determined neutralization titer. The final preparation contained 10,000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins in a single volume of 10 ml. T-BAT preparations were then stored in separate packages at 4 °C for further assays.

Potency assay of T-BAT

To study the preventive effect of T-BAT against BoNT challenge, KM mice (18–22 g, n = 8 per sub-group) were injected intravenously with 10, 40, or 100 μl T-BAT. At 1, 3, 5, or 8 days later, the mice were then injected intraperitoneally with different doses of mixed BoNTs (i.e., mixed toxin containing 5, 20, 100, or 500 LD50 of each BoNT). Survival of the mice was monitored for 2 weeks.

To evaluate the therapeutic effect of T-BAT on post-exposure to BoNTs, KM mice (18–22 g, n = 8 per sub-group) were injected intraperitoneally with various amounts of each BoNT (i.e., mixed toxins containing 0.25, 1, 5, or 20 LD50 of each BoNT), and then T-BAT (10, 40, or 100 μl) was injected intravenously at 1, 3, or 6 h later. Survival of the mice was monitored for 2 weeks.

Acute toxicity assessments of the T-BAT

Acute toxicity analysis was performed on each batch of T-BAT following the guidelines of the Pharmacopoeia of the People’s Republic of China (PPRC, Appendix XII F: Abnormal toxicity screening method). Briefly, 10 mice (18–22 g) were randomized into two groups, which received a single injection of T-BAT or saline as a control. The injected dose was 500 μl of T-BAT per mouse. The mice were then observed for 2 weeks, during which time we recorded their clinical status, such as pathology, food consumption, and body weight.

Statistical analysis

The mean ± the standard deviation was used to express the quantitative data. One-way analysis of variation (ANOVA) for multiple comparisons and Student’s t-test for pairwise comparisons were used to analyze the data statistically. We used Fisher’s exact test to assess the statistical differences for survival between the groups. For all the statistical tests, only those data that resulted in a P value < 0.05 were considered statistically significant.

Results

Pilot-scale production and characterization of recombinant BoNT antigens

Pilot-scale production of recombinant BoNT antigens was established as described previously (Shi et al. 2022). The information on these antigens are listed in Supplementary Table S1. The non-His-tagged recombinant BoNT/A, BoNT/E, and BoNT/F Hc antigens (AHc, EHc, and FHc) were expressed in E. coli, and the recombinant Hc antigen of BoNT/B was expressed in yeast. Validated antigen batches (Supplementary Table S2) were used to immunize horses.

Preparation of horse sera with high antibody titers

The horses were immunized with recombinant antigens, and their sera with high antibody titer were collected. After a predetermined number of immunizations, the sera antibody titer and neutralization potency were measured immediately. The results (Fig. 1) showed that as the number of immunizations increased, the antibody titers gradually increased. After six immunizations, the antibody titer of horse sera reached a high level, averaging over 1:409,600. The antibody titer was maintained at a high level after further immunization, reaching more than 1:409,600 after the eighth and ninth immunizations, indicating the good immunogenicity of the pilot-scale recombinant antigens and that they could produce high neutralizing antibody levels after immunization.

Fig. 1.

Fig. 1

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Antibody titer assay of horse sera. ELISA was used to measure the anti-RBD antibody titers of the hyperimmune sera. A Anti-AHc antibody titer, B anti-BHc antibody titer, C anti-EHc antibody titer, and D anti-FHc antibody titer

The neutralization potencies of the horse sera are listed in Table 1. If the criteria of neutralization antibody were met (3000 IU/ml for serotype A, 2000 IU/ml for serotype B and F, and 1000 IU/ml for serotype E, respectively), then a bulk blood collection was performed on these horses and the neutralizing potency was again tested. The results showed that 60–80% of these horses were able to reach high potency, indicating that the recombinant immunization protocol was very efficient. Qualified horse sera were stored at 4 °C for further antitoxin production. In conclusion, we established an effective immunization program and obtained a total of 342 L of serotype A, 240 L of serotype B, 134 L of serotype E, and 210 L of serotype F high potency horse sera.

Table 1.

The neutralization potency of serum from horses immunized with various serotypes of BoNT recombinant antigens

Number of immunization Neutralization potency (IU/ml)
Serotype A Mean (SD) Serotype B Mean (SD) Serotype E Mean (SD) Serotype F Mean (SD)
4 2433 812 1971 750 330 242 2167 236
5 3575 1107 2550 1452 810 623 2500 866
6 4429 1208 2406 984 1560 571 2750 829
7 4167 687 3000 707 1750 1137 3714 1578
8 4250 1785 2667 1067 2600 1200 4500 1118
9 4500 1541 3500 764 2909 1505 4000 2236
10 4694 1761 3944 1423 3450 1524 4333 2134
11 5167 850 4278 1133 4000 1466 4000 2363
12 5083 932 4350 1074 4375 1192 4333 2134
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Preparation and characterization of monovalent antitoxins

Two batches of monovalent antitoxins against the four BoNTs were prepared and characterized in this study. The SDS-PAGE analysis of these antitoxins (Fig. 2) showed that the purified F(ab′)2 product migrated as an approximately 100-kDa band. The purity of F(ab′)2 product was > 77.7% (Table 2), without IgG, based on the results of non-reduce SDS-PAGE. Their neutralization antibody levels reached above 4000 IU/ml, indicating that the M-BATs were of high efficacy and high quality. Notably, the BoNT/B antitoxin was produced using yeast-expressed Hc antigen, suggesting that recombinant proteins derived from the yeast can be used as effective antigens to develop vaccine or antitoxins.

Fig. 2.

Fig. 2

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Analysis of antitoxins with non-reduce SDS-PAGE. Lines: 1–2, BoNT/A antitoxin. Lines: 3–4, BoNT/B antitoxin. Lines: 5–6, BoNT/F antitoxin. Lines: 7–8, BoNT/E antitoxin. M, protein markers. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Arrow indicates the F(ab′)2 (about 100 kDa) in figure

Table 2.

Characterization of monovalent F(ab′)2 antitoxins

Item Standard Results
A01 A02 B01 B02 E01 E02 F01 F02
pH 6.0–7.0 6.6 6.7 6.6 6.6 6.7 6.6 6.6 6.7
Identification experiment Positive reaction with equine IgG antibody Positive Positive Positive Positive Positive Positive Positive Positive
Neutralization potency (IU/ml)  ≥ 2000 7000 5000 4000 6000 4000 6000 4000 4000
F(ab')2 (%)  ≥ 70 77.7 79.5 81.4 83.2 85.6 83.5 78.9 80.2
Protein content (%)  ≤ 170 mg/ml 68.2 54.6 62.3 82.1 65.8 80.4 59.7 60.3
Specific activity  ≥ 50 IU/mg 103.0 91.6 64.2 73.1 60.8 74.6 67.0 66.3
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The immunological characterization of anti-BoNT toxoid and anti-Hc horse sera antibodies

In this study, we explored the immunological characteristics of anti-BoNT toxoid and anti-Hc horse sera antibodies. As shown in supplemental Figure S1, anti-BoNT/A toxoid horse sera antibodies could bind to all of domains derived from AL-HN of BoNT/A with a strong binding affinity (Supplementary Fig. S1C), but bound weakly to the AHc and AHc-N domains derived from the RBD of BoNT/A and not at all to the AHc-C domain (Supplementary Fig. S1B). Moreover, anti-AHc horse sera antibodies bound specifically to all domains derived from the RBD of BoNT/A (Supplementary Fig. S1D), but not to all of domains derived from AL-HN of BoNT/A (Supplementary Fig. S1E). Therefore, there are more abundant specific-L-HN antibodies, but no specific-AHc-C domain antibodies, in the anti-BoNT/A toxoid horse sera antibodies.

Anti-BoNT/B toxoid horse sera antibodies showed strong binding affinities to both BHc and BL-HN domains (Supplementary Fig. S2B), but bound weakly bind to the BHc-N domain and not at all to the BHc-C domain (Supplementary Fig. S2D). As expected, anti-BHc horse antibodies bound specifically to the BHc domain, but not to the BL-HN domain (Supplementary Fig. S2C).

Anti-BoNT/E toxoid horse sera antibodies showed strong binding affinities for all domains derived from EL-HN of BoNT/E (Supplementary Fig. S3B), but bound weakly to EHc and EHc-N domains derived from the RBD of BoNT/E and could not bind to the EHc-C domain (Supplementary Fig. S3B).

Anti-BoNT/F toxoid sera horse antibodies could bind to all of domains derived from FL-HN of BoNT/F with a strong binding affinity, but could not bind to the FHc domain (Supplementary Fig. S4A). As expected, anti-FHc horse sera antibodies could specifically bind to all of domains derived from the RBD of BoNT/F (Supplementary Fig. S4B).

The immunological characterizations of anti-BoNT toxoid and anti-Hc horse sera antibodies on different domains of BoNTs were also confirmed using ELISA, and the similar binding characterizations as these western blot experiments were observed. In summary, according to the specific antigen–antibody binding characteristics, there are more abundant specific-L-HN antibodies, no or low specific-Hc-C domain or relative weak specific-RBD antibodies, in the anti-BoNT toxoid antibodies. Based on the Hc-C domain as the double receptor binding domain of toxin-receptor action, these specific-Hc-C or RBD antibodies should play more important roles in inhibiting toxin binding to the receptors on the neuronal cell surface.

No cross-protection of certain M-BAT against other BoNTs

BoNTs are classified into seven serotypes (A–G), with differences of up to 63% at the amino acid level, which show little antibody cross-reactivity. In this study, the cross-protection of certain botulinum antitoxin against other BoNTs was explored using a monovalent antitoxin based on the Hc domain or toxoid. The A serotype monovalent antitoxin was diluted from 3.75 to 960 IU/ml, then mixed with 1 L + /ml (I.e., 10 000 LD50/ml) of each neurotoxin serotype B, E, and F. Mice were protected against the homologous neurotoxin (i.e., anti-A serotype antitoxin protected against BoNT/A), but protection against other neurotoxin serotypes was not observed up to an antitoxin to toxin a ratio of 960:1 (Table 3). Our result also proved that the antitoxins of serotype A based on the Hc domain or toxoid produced no cross-protection against other BoNTs (Li et al. 2012).

Table 3.

Neutralization capacity of monovalent serotype A antitoxin based on the toxoid or Hc domain for other serotype botulinum neurotoxins (BoNTs)

Antitoxin Serotype of BoNTs Ratio of antitoxin/toxina
3.75:1 7.5:1 15:1 30:1 60:1 120:1 240:1 480:1 960:1
Monovalent serotype A antitoxin based on toxoidb A 4/4c 4/4 /d / / / / / /
B 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
E 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
F 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
Monovalent serotype A antitoxin based on Hc domainb A 4/4c 4/4 /d / / / / / /
B 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
E 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
F 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
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aIn this experiment, the monovalent antitoxin of serotype A was mixed with the same dose of each BoNT (serotypes A, B, E, and F). According to neutralization potency of monovalent serotype A antitoxin, ratios of antitoxin /toxin were designed from 3.75:1 to 960:1 for each BoNT

bMonovalent antitoxin of serotype A based the toxoid is from Lanzhou Institute of Biological Products Co., Ltd. Monovalent antitoxin of serotype A based on Hc domain is prepared in this study

cIn each sub group, 4 mice are injected to assess neutralization potency of monovalent antitoxin of serotype A against each BoNT

dNot done. The protection against BoNT/A could be observed with an antitoxin to toxin a ratio of 1:1

In addition, the M-BATs of other serotypes (B, E, and F) showed no cross-reactive immunoprotective effect. Our result proved that none of the antitoxins produced cross-protection against other BoNTs. Therefore, tetravalent botulinum antitoxins (T-BATs) should been prepared to protect against each BoNT related to human botulism.

Preparation and characterization of T-BATs

Two batches of T-BAT were prepared and named as T-BAT01 and T-BAT02. Characterization of the T-BATs was then carried out. The results showed that the T-BATs could protect the mice against the four serotypes of BoNT and the potency of both T-BATs reached our expectations (Supplemental Table S3). Both T-BAT01 and T-BAT02 with a 10-ml volume contained at least 10,000 IU of BoNT/A and at least 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins.

Under non-reducing conditions, the F(ab′)2 in T-BATs migrated as a single 100 kDa band, whereas under reducing conditions, they migrated as 25–31-kDa bands (Fig. 3), indicating that digestion of the immunoglobulins was complete, because no undigested heavy chains were seen under reducing conditions. The purity of the T-BATs was > 80%. Besides, the T-BATs remained stable and potent when stored at 4 °C for up to 2 years (Fig. 3C–D).

Fig. 3.

Fig. 3

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Analysis of T-BAT under non-reducing (A, C, and D) and reducing (B) conditions using SDS-PAGE. In all figures, line 1, T-BAT01; line 2, T-BAT02. M, protein markers; T-BATs were stored at 4 °C for 1 year (C) or 2 years (D). Arrows indicate H and L of F(ab′)2 in figure B. Arrow indicates the F(ab′)2 (about 100 kDa) in figure D

Pharmacodynamic evaluation of T-BAT

To further evaluate the pharmacodynamic potency of T-BAT, in vivo prevention and treatment experiments were performed in a mouse model. In the in vivo prevention experiment, 10, 40, and 100 μl of T-BAT were administered by tail vein to each mouse. Then, the mice were challenged within 1–8 days with mixed neurotoxins containing 5, 20, 100, or 500 LD50 of each BoNT. The results (Table 4) showed that T-BAT has a strong ability to prevent poisoning by various serotypes of botulinum neurotoxin, and the preventive effect was dose- and time-dependent. Administration of 100 μl of antitoxin completely protected against 100 LD50 mixed neurotoxin challenge on day 5, and partially protected against 5 LD50 mixed toxins challenge on day 8. In the low-dose group, 10 μl of antitoxin could also provide complete protection against 100 LD50 of mixed neurotoxins within 3 days. These results show that T-BAT provides a good protective effect in a short period, and can be used to prevent of poisoning by various serotypes of botulinum neurotoxin in emergency.

Table 4.

Preventive effects of T-BAT against mixed BoNTs in mice

T-BAT (μl) Survival (number alive/8 tested)
5 LD50 20 LD50 100 LD50 500 LD50
1d 3d 5d 8d 1d 3d 5d 8d 1d 3d 5d 8d 1d 3d 5d 8d
10 8 8 6 0 8 8 0 0 8 8 0 0 8 0 0 0
40 8 8 8 0 8 8 8 0 8 8 4 0 8 8 0 0
100 8 8 8 4 8 8 8 0 8 8 8 0 8 8 4 0
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10, 40, and 100 μl of T-BAT01, which equated to 19.65, 78.6, and 196.5 mg/kg, were used this study. The mice weighed 20 g, and the human weight was assumed to be 60 kg, giving a weight ratio of 3000. The above doses are equivalent to 3, 12, and 30 times of the human dose, respectively. However, the mixed neurotoxins were used in the doses up to 100 times of 50% mouse lethal dose. In this experiment, 10, 40, and 100 μl of T-BAT01 was administered intraperitoneally to each mouse. Then, the mice (8 mice/group) were challenged within 1, 3, 5, or 8 days with mixed neurotoxins containing 5, 20, 100, or 500 LD50 of each BoNT

In the in vivo treatment experiment, mice were injected i.p. with mixed BoNTs containing 0.25, 1, 5, or 20 LD50 of each BoNT. Then, 10, 40, or 100 μl of T-BAT was injected by tail vein for treatment within 1–6 h, respectively. The results showed that after the mice were injected with the mixed neurotoxin, the symptoms of poisoning became obvious, and the mice died in large numbers after 6 h. However, T-BAT could effectively treat poisoning by the four serotype botulinum neurotoxins, in a time- and dose-dependent manner. For the 20 LD50 challenge, mice could only be partly saved by 40 or 100 μl of T-BAT treatment. For the 5 LD50 challenge group, the mice could be successfully saved by all doses of T-BAT at 1 h, and were partly treated by a high dose T-BAT (40 μl) after 3 h. For the 1 LD50 challenge, the mice could be saved within 3 h by all doses of T-BAT, but only partly survived when treated 6 h after challenge. However, for the 0.25 LD50 of each BoNT challenge, mice could be cured by T-BAT at all times (Table 5). In contrast, in the prevention and treatment experiments, pre-immune equine F(ab′)2 control injection provided no protection for the mice against BoNT challenge. The result indicated that T-BAT could neutralize all four serotypes of BoNTs at the same time, delayed the poisoning process, and reduced poisoning symptoms, thereby achieving successful treatment of botulism.

Table 5.

Therapeutic effects of T-BAT against mixed BoNTs in mice

BoNTs (LD50) Survival (number alive/8 tested)
1 h 3 h 6 h
10 μl 40 μl 100μl 10 μl 40 μl 100 μl 10 μl 40 μl 100 μl
0.25 8 8 8 8 8 8 8 8 8
1 8 8 8 8 8 8 0 4 6
5 8 8 8 0 3 4 0 0 0
20 0 4 6 0 0 0 0 0 0
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In this experiment, mice (8 mice/group) were injected intraperitoneally with mixed BoNTs containing 0.25, 1, 5, or 20 LD50 of each BoNT. Then, 10, 40, and 100 μl of T-BAT01 was injected via the tail vein for treatment within 1, 3, or 6 h, respectively. The mixed neurotoxins were used in the doses up to 20 times of 50% mouse lethal dose. Similar results were observed for the in vivo prevention and treatment experiments using T-BAT02

Acute toxicity test of T-BAT

The acute toxicity of T-ABT was tested in as preclinical trail according to the Pharmacopoeia of the People’s Republic of China (PPRC, Appendix XII F). During the observation period, all mice remained healthy and did not react abnormally. After 7 days of observation, the body weight of each mouse increased significantly (Supplemental Fig. S5; p < 0.001), indicating that the tetravalent antitoxin sample passed the acute toxicity test.

Discussion

The highly toxic botulinum neurotoxins (BoNTs) have been developed as biological warfare agents by many countries and organizations. Vaccination and antitoxins are the only efficient ways to prevent and treat botulism. At present, these researches to develop new botulinum vaccines have focused on the non-toxic recombinant BoNTs, such as Hc, which is expressed in E. coli (Baldwin et al. 2008; Ben David et al. 2022; Gao et al. 2010; Moreira et al. 2014; Shi et al. 2019; Webb et al. 2017), yeast (Liu et al. 2015; Sinha et al. 2007; Smith 2009), or 293E cell (Yu et al. 2015). Similarly, we have prepared monovalent botulism antitoxins against BoNT A, E, and F using recombinant Hc antigens expressed in E. coli (Shi et al. 2020; Yu et al. 2010a, 2010c).

Herein, a novel tetravalent botulism antitoxin (T-BAT) was developed that could prevent poisoning with human botulism-inducing serotypes of A, B, E, and F simultaneously. Firstly, we verified that the yeast-expressed recombinant Hc antigen of BoNT/B elicited comparative antibody potency compared with those expressed in E. coli (Gao et al. 2010; Liu et al. 2015; Shi et al. 2019). Horse sera of high potency was used successfully to prepare a monovalent BoNT/B antitoxin with high quality and neutralizing potency. These results verified that the yeast expression system can also be used for antitoxin immunogen preparation, thus expanding the applicability of this novel antitoxin-preparation method.

The Hc of BoNT is also one of the most widely sequence divergent domains of the BoNT protein. The lack of apparent cross protection between the based-RBD antitoxin and other heterologous BoNTs was observed in current experimental conditions. The monovalent BoNT/B antitoxin, along with the other three monovalent antitoxins, was used to prepare T-BAT. After the characterization of T-BAT preparations, the in vivo prevention and treatment experiments were performed in a mouse model. The therapeutic efficacy of H-BAT against each BoNT has been proven in different animal models (Barker et al. 2019; Emanuel et al. 2019; Kodihalli et al. 2017), and our results showed that T-BAT has the ability to neutralize four serotypes of BoNTs in the mice model. More importantly, our results demonstrated that T-BAT has strong therapeutic efficacy against challenge with four serotypes of BoNTs simultaneously, and delayed the poisoning process and reduced poisoning symptoms, achieving successful treatment of botulism. This is the first novel T-BAT preparations to successfully protect against four serotypes of BoNTs simultaneously. The results validated that the protocol for preparing tetravalent antitoxins is suitable to prepare multivalent antitoxins, including other toxins, such as tetanus.

The T-BAT produced in this study is based on RBD of the neurotoxins as immunogen. T-BAT can specifically bind the RBD, preventing it from binding to the receptor region and accelerating its metabolism, thus achieving neutralization and removal of BoNTs, which is very important in the prevention of BoNT poisoning, because the neutralization effect worked before the entry of neurotoxins into nerve cells in the body. In fact, our results further proved that the T-BAT could neutralize recombinant chimeric BoNT E/A or A/E (Nugent et al. 2018; Wang et al. 2008), because the T-BAT was able to bind to all the RBDs of the four BoNTs. In addition, we found that most of the neutralizing antibodies in the based-inactivated toxoid monovalent antitoxin (produced by Lanzhou Institute of Biological Products Co., Ltd, LanZhou, China) targeted the non-receptor binding domain (L-HN), whereas only a small part of the neutralizing antibody targeted RBD (Data not shown), which was consistent with the immunological characterization of anti-BoNT toxoid and anti-Hc horse sera antibodies on the domains of BoNTs. In our previous studies (Li et al. 2021, 2022, 2023; Liu et al. 2020), the L-HN proteins of BoNT/A and B are found to induce sufficient protection as the their Hc domains and the L-HN proteins of BoNT/E and F had a better immune protection effect and neutralizing antibody response than the their Hc domains. These results suggested that the effective neutralizing antibodies are more concentrated in RBD epitopes in the novel T-BAT, while toxoid-based antitoxins are more concentrated in L-HN epitopes, resulting in more effective treatment by T-BAT than by traditional antitoxins.

In addition, the specific activity of the anti-AHc antitoxin in our study was fourfold higher that of the anti-toxoid antitoxins (Lanzhou Institute of Biological Products Co., Ltd). This result also indicated that the neutralizing effects of the anti-Hc antibodies were more potent than those of anti-toxoid antitoxins (Ben David et al. 2022), which is related to the core neutralizing epitopes in the RBD. This novel tetravalent botulinum antitoxin based on the RBD of BoNTs could effectively treat botulism caused by BoNT/A, B, E, and F. We also hypothesized that it could neutralize all recombinant chimeric neurotoxins containing the RBD of BoNT/A, B, E, and F.

In conclusion, we successfully developed a novel T-BATs that could treat or prevent BoNT/A, B, E, and F simultaneously using the BoNT Hc structural domains as antigens. These results demonstrated that the yeast-expressed antigen (BHc) was equally efficacious in eliciting an effective antitoxin. The findings of the present study experimentally validated RBD-specific novel antitoxins as pharmaceutical anti-botulinum equine antitoxins to treat BoNT/A-, B-, E-, and F-induced botulism. However, further characterization of the efficacy and pre-clinical safety of T-BAT is required, after which, clinical trials of this novel BoNT antitoxin in humans can be initiated. Herein, we further demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of the toxin as an alternative to inactivated toxins.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 727 KB) (726.5KB, pdf)

Author contribution

Conceived and designed the experiments: YZY and ZXY. Performed the experiments: DYS, JSL, YYM, FJL, and RW. Analyzed the data: DYS, YZY, JSL, and FJL. Contributed reagents/materials/analysis tools: YYM, RW, PD, and SY. Wrote the paper: DYS, YZY, and JSL.

Funding

This work was partly supported by the Biosafety Research Project (grant number 20SWAQX23-002–002) and Laboratory of Advanced Biotechnology Project (grant number BDZZ202204).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

The Animal Care and Use Committee of the Beijing Institute of Biotechnology approved all the animal experiments, which were carried out following the institute’s ethical guidelines and the National Institutes of Health guide for the care and use of laboratory animals (IACUC-DWZX-2019–017).

Conflict of interest

The authors declare no competing interests.

Disclaimer

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Dan-Yang Shi, Jian-Sheng Lu, Yun-Yun Mao contributed equally to this work.

Contributor Information

Yun-Zhou Yu, Email: yunzhouyu@163.com.

Zhi-Xin Yang, Email: yy_xiao@126.com.

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Associated Data

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Supplementary Materials

Supplementary file1 (PDF 727 KB) (726.5KB, pdf)

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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