# Complete Neutralization of Tetanus Neurotoxin by Alpaca-Derived Trivalent Nanobodies Aimed at Veterinary Medical Applications

## TL;DR

Researchers developed a powerful alpaca-derived antibody that neutralizes tetanus toxin more effectively than current treatments, offering a sustainable solution for both human and animal use.

## Contribution

A trivalent alpaca-derived nanobody with 1580 IU/mg neutralizing activity against tetanus neurotoxin, surpassing existing antitoxins.

## Key findings

- tVHH-8/11/36 neutralized tetanus neurotoxin with 1580 IU/mg activity, exceeding human and veterinary immunoglobulin preparations.
- The trivalent VHH binds multiple toxin domains, creating synergistic neutralization through multimerization.
- The technology enables microbial production, addressing animal welfare and manufacturing efficiency.

## Abstract

Tetanus causes fatal neurological diseases in both humans and animals. In this study, we developed an alpaca-derived trimer, VHH, and achieved high neutralizing activity against tetanus neurotoxin. The tVHH-8/11/36, which links three selected nanobodies, demonstrated a neutralizing activity of approximately 1580 IU/mg, greatly exceeding that of human and veterinary anti-tetanus immunoglobulin preparations. Trimer VHH can be mass-produced in microorganisms, achieving both animal welfare considerations and manufacturing efficiency. This technology is expected to serve as a next-generation antitoxin that embodies the One Health concept, bridging veterinary and human medicine.

Tetanus is a zoonotic disease posing significant threats to both humans and animals, particularly horses, sheep, and ruminants. Current antitoxin therapies rely on animal-derived immunoglobulins, presenting challenges including animal welfare concerns, pathogen contamination risks, and manufacturing complexity. Alpaca-derived nanobodies (VHH) are promising alternatives owing to their high antigen-binding affinity, thermostability, and potential for microbial production. We developed highly active trivalent VHH antibodies (tVHH) that target multiple epitopes of tetanus neurotoxin (TeNT). Following alpaca immunization with tetanus toxoid, 41 VHH clones were isolated using phage display. Six VHH clones were selected through in vivo neutralization assays, from which three clones of VHH (8, 11, 36) were selected to construct tVHH-8/11/36 and tVHH-8/36/11. Using an improved 21-day mouse neutralization assay, tVHH-8/11/36 demonstrated exceptional neutralizing activity of approximately 1580 IU/mg against 4000 LD50 of toxin, substantially exceeding current human and veterinary anti-tetanus immunoglobulin preparations. Surface plasmon resonance and ELISA confirmed that each VHH recognizes different TeNT domains, producing synergistic neutralizing effects through multimerization. Since antitoxin therapy challenges are common to both animals and humans, this tVHH technology supports One Health by providing a unified therapeutic platform applicable across species through sustainable microbial production.