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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.

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.

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.