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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.

Tetanus toxin (TeNT) is one of the most toxic proteins. Neutralizing antibodies against TeNT are effective in prevention and treatment. In this study, 14 anti-tetanus nanobodies were obtained from a phage display nanobody library by immunizing a camel with the C-terminal receptor-binding domain of TeNT (TeNT-Hc) as the antigen. After fusion with the human Fc fragment, 11 chimeric heavy-chain antibodies demonstrated nanomolar binding toward TeNT-Hc. The results of toxin neutralization experiments showed that T83–7, T83–8, and T83–13 completely protected mice against 20 × the median lethal dose (LD50) at a low concentration. The neutralizing potency of T83–7, T83–8, and T83–13 against TeNT is 0.4 IU/mg, 0.4 IU/mg and 0.2 IU/mg, respectively. In the prophylactic setting, we found that 5 mg/kg of T83–13 provided the mice with full protection from tetanus, even when they were injected 14 days before exposure to 20 × LD50 TeNT. T83–7 and T83–8 were less effective, being fully protective only when challenged 7 or 10 days before exposure, respectively. In the therapeutic setting, 12 h after exposure to TeNT, 1 ~ 5 mg/kg of T83–7, and T83–8 could provide complete protection for mice against 5 × LD50 TeNT, while 1 mg/kg T83–13 could provide complete protection 24 h after exposure to 5 × LD50 TeNT. Our results suggested that these antibodies represent prophylactic and therapeutic activities against TeNT in a mouse model. The T83–7, T83–8, and T83–13 could form the basis for the subsequent development of drugs to treat TeNT toxicity.

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.

BackgroundBotulinum 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.MethodsHerein, 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.ResultsThe 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.ConclusionThe 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.

Abstract Botulinum neurotoxin (BoNT) shows high lethality and toxicity, marking it as an important biological threat. The only effective post-exposure therapy is botulinum antitoxin; however, such products have great potential for improvement. To prevent or treat BoNT, monoclonal antibodies (mAbs) are promising agents. Herein, we aimed to construct a bispecific antibody (termed LUZ-A1-A3) based on the anti-BoNT/A human monoclonal antibodies (HMAb) A1 and A3. LUZ-A1-A3 binds to the Hc and L-HN domains of BoNT/A, displaying potent neutralization activity against BoNT/A (124 × higher than that of HMAb A1 or HMAb A3 alone and 15 × higher than that of the A1 + A3 combination). LUZ-A1-A3 provided effective protection against BoNT/A in an in vivo mouse model. Mice were protected from infection with 500 × LD50 of BoNT/A by LUZ-A1-A3 from up to 7 days before intraperitoneal administration of BoNT/A. We also demonstrated the effective therapeutic capacity of LUZ-A1-A3 against BoNT/A in a mouse model. LUZ-A1-A3 (5 μg/mouse) neutralized 20 × LD50 of BoNT/A at 3 h after intraperitoneal BoNT/A administration and complete neutralized 20 × LD50 of BoNT/A at 0.5 h after intraperitoneal BoNT/A administration. Thus, LUZ-A1-A3 is a promising agent for the pre-exposure prophylaxis and post-exposure treatment of BoNT/A.

BackgroundHuman adenovirus type 55 (HAdV55) has a re-emerged as pathogen causing an acute respiratory disease presenting as a severe lower respiratory illness that can cause death. To date, there is no HAdV55 vaccine or treatment available for general use.MethodsHerein, a monoclonal antibody specific for HAdV55, mAb 9-8, was isolated from an scFv-phage display library derived from mice immunized with the purified inactived-HAdV55 virions. By using ELISA and a virus micro-neutralization assay, we evaluated the binding and neutralizing activity of mAb 9-8 following humanization. Western blotting analysis and antigen-antibody molecular docking analysis were used to identify the antigenic epitopes that the humanized monoclonal antibody 9-8-h2 recognized. After that, their thermal stability was determined.ResultsMAb 9-8 showed potent neutralization activity against HAdV55. After humanization, the humanized neutralizing monoclonal antibody (9-8-h2) was identified to neutralize HAdV55 infection with an IC50 of 0.6050 nM. The mAb 9-8-h2 recognized HAdV55 and HAdV7 virus particles, but not HAdV4 particles. Although mAb 9-8-h2 could recognize HAdV7, it could not neutralize HAdV7. Furthermore, mAb 9-8-h2 recognized a conformational neutralization epitope of the fiber protein and the crucial amino acid residues (Arg 288, Asp 157, and Asn 200) were identified. MAb 9-8-h2 also showed favorable general physicochemical properties, including good thermostability and pH stability.ConclusionsOverall, mAb 9-8-h2 might be a promising molecule for the prevention and treatment of HAdV55.

Botulinum toxin A(BoNT/A) is a neurotoxin produced by the bacteria Clostridium botulinum, which can cause serious food poisoning and is recognized as a potential biological warfare agent. BoNT/A is does not degrade easily and can remain in the complex matrix for a long time. Meanwhile, the poisonous dose of botulinum toxin exceptionally low and intravenous human lethal doses estimated at 1-3 ng/kg. Therefore, sensitive and accurate detection methods suitable for testing a wide range of complex samples are urgently needed. To this end, the “amplified luminescent proximity homogeneous assay linked immunosorbent assay” (AlphaLISA) was established for the detection of BoNT/A and its detection efficacy in plasma, beverage, food, and other complex samples was evaluated. The results showed that this method can very effectively resist matrix interference. The detection time is rapid, reaching a detection limit for all samples of up to 0.1 ng/mL in only 30 min. BoNT/A can also be accurately detected in vomit samples of patients with clinical food poisoning. This study demonstrates that AlphaLISA is an effective tool for the detection of BoNT/A in complex samples and can potentially be developed for commercial use in the future.

Abstract Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is generally known to be the most poisonous of all biological toxins. In this study, we evaluate the protection conferred by intratracheal (i.t.) inoculation immunization with recombinant Hc subunit (AHc) vaccines against aerosolized BoNT/A intoxication. Three AHc vaccine formulations, i.e., conventional liquid, dry powder produced by spray freeze drying, and AHc dry powder reconstituted in water are prepared, and mice are immunized via i.t. inoculation or subcutaneous (s.c.) injection. Compared with s.c.-AHc-immunized mice, i.t.-AHc-immunized mice exhibit a slightly stronger protection against a challenge with 30,000× LD50 aerosolized BoNT/A. Of note, only i.t.-AHc induces a significantly higher level of toxin-neutralizing mucosal secretory IgA (SIgA) production in the bronchoalveolar lavage of mice. In conclusion, our study demonstrates that the immune protection conferred by the three formulations of AHc is comparable, while i.t. immunization of AHc is superior to s.c. immunization against aerosolized BoNT/A intoxication.

Botulinum neurotoxins (BoNTs) can cause nerve paralysis syndrome in mammals and other vertebrates. BoNTs are the most toxic biotoxins known and are classified as Class A biological warfare agents. BoNTs are mainly divided into seven serotypes A-G and new neurotoxins BoNT/H and BoNT/X, which have similar functions. BoNT proteins are 150 kDa polypeptide consisting of two chains and three domains: the light chain (L, catalytic domain, 50 kDa) and the heavy chain (H, 100 kDa), which can be divided into an N-terminal membrane translocation domain (HN, 50 kDa) and a C-terminal receptor binding domain (Hc, 50 kDa). In current study, we explored the immunoprotective efficacy of each functional molecule of BoNT/F and the biological characteristics of the light chain-heavy N-terminal domain (FL-HN). The two structure forms of FL-HN (i.e., FL-HN-SC: single chain FL-HN and FL-HN-DC: di-chain FL-HN) were developed and identified. FL-HN-SC could cleave the vesicle associated membrane protein 2 (VAMP2) substrate protein in vitro as FL-HN-DC or FL. While only FL-HN-DC had neurotoxicity and could enter neuro-2a cells to cleave VAMP2. Our results showed that the FL-HN-SC had a better immune protection effect than the Hc of BoNT/F (FHc), which indicated that L-HN-SC, as an antigen, provided the strongest protective effects against BoNT/F among all the tested functional molecules. Further in-depth research on the different molecular forms of FL-HN suggested that there were some important antibody epitopes at the L-HN junction of BoNT/F. Thus, FL-HN-SC could be used as a subunit vaccine to replace the FHc subunit vaccine and/or toxoid vaccine, and to develop antibody immune molecules targeting L and HN domains rather than the FHc domain. FL-HN-DC could be used as a new functional molecule to evaluate and explore the structure and activity of toxin molecules. Further exploration of the biological activity and molecular mechanism of the functional FL-HN or BoNT/F is warranted.

Background: As a Class A bioterrorism agent, botulinum neurotoxin serotype A (BoNT/A) carries the risk of being used by terrorists to cause mass poisoning. The microneedle (MN) patch has a great potential for application as a novel vaccine delivery method. The aim of this study is to develop a thermally stable, dissolving microneedle patch for the delivery of a recombinant protein vaccine using a recombinant C-terminal heavy chain of BoNT/A (Hc of BoNT/A, AHc) to prevent botulism. Methods: Fish gelatin, a natural non-toxic and bacteriostatic material, was selected as the microneedle matrix for the preparation of the dissolving microneedle vaccine. Subsequently, the mechanical performance, bacteriostatic properties, vaccination effect, and stability of the microneedle patches were evaluated using instruments such as the displacement-force test station and optical coherence tomography (OCT) scanner. Results: Fish gelatin matrix at high concentrations has good bacteriostatic properties, and excellent mechanical performance and vaccination effect, meeting the necessities of a vaccine. In both in vivo and in vitro neutralization experiments, MN vaccines containing different antigen doses achieved the same protective efficacy as subcutaneous vaccinations, protecting mice against 106 LD50 of BoNT/A injected intraperitoneally. Thermal stability analysis of the MN vaccines revealed that the fish gelatin matrix protected the AHc vaccine from protein denaturation even after 7 days of storage at 37 °C and enabled the vaccine patches to maintain good immunogenicity and protective efficacy even after 6 months of storage at room temperature. Conclusion: In this study, we successfully prepared a bacteriostatic MN patch using a fish gelatin matrix that not only has a good vaccination effect, but also obviates the need for a cold chain for the AHc vaccine, providing the possibility of rapid, painless, and large-scale vaccination.

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure–activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2–3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9–12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of “1–4, 2–3” (ribbon), which differs from the “1–3, 2–4” (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity “1–4, 2–3,” are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.

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.

B1-type human adenoviruses (HAdVs) HAdV-3, HAdV-7, and HAdV-55 have caused epidemics in North America, Asia, and Europe. However, to date, no adenovirus vaccines or antiviral drugs have been approved for general use. In the present work, a scFv-phage immune library was constructed and mouse monoclonal antibody (MMAb) 10G12 was obtained through selection. 10G12 is specific for HAdV-7 and binds the hexon loop1 and loop2 (LP12), resulting in strong neutralization activity against HAdV-7. Additionally, it is stable in serum and buffer at various pH values. The findings provide insight into adenovirus and antibody responses and may facilitate the design and development of adenovirus vaccines and antiviral drugs.

To improve efficacy and minimize toxicity of EGFR inhibition treatment, we developed Ame55, a novel anti-EGFR IgG1 with lower affinity to EGFR than cetuximab (C225) from a human phage library. Ame55 had lower bioactivity than cetuximab in vitro but similar antitumor efficacy as cetuximab in vivo. Moreover, Ame55 was more efficacious than cetuximab in a Lovo cell xenograft tumor model when combined with irinotecan (CPT-11). Ame55 concentrates in the mouse xenograft tumor and has less toxicity than cetuximab in cynomolgus monkeys in an overdose study.

The incidence of dengue virus (DENV) infections has been escalating in tropical and subtropical countries, but there are still no effective therapeutic options. In the present study, a DENV-1-specific human monoclonal antibody (HMAb), 1G5, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of dengue patients was found to have potent neutralization activity against serotype 1 DENV (DENV-1). Its neutralization activity against DENV-2 was not as strong, and it was almost absent for DENV-3 and DENV-4. The results showed that HMAb 1G5 only binds to the envelop protein of intact DENV-1 or the envelop protein under unheated and non-reducing conditions, and that it does not bind to recombinant envelope protein. This could mean that the antibody recognizes a conformational epitope of the envelope protein. Further, the findings showed that HMAb 1G5 potently neutralizes DENV-1 in both the pre- and post-attachment phases of the virus at low concentrations. In vivo studies showed that HMAb 1G5 provides protection from DENV-1 infection in a murine model. In addition, antibody-dependent enhancement that occurs at lower doses of the antibody was completely abrogated by the introduction of Leu-to-Ala mutations (1G5-LALA) or deletion of nine amino acids (1G5-9del) in the Fc region. Therefore, HMAb 1G5 shows promise as a safe and effective agent for prophylactic and therapeutic treatment of DENV-1 infection.