{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T10:00:15Z","timestamp":1792058415705,"version":"4.4.0"},"reference-count":23,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,19]],"date-time":"2026-01-19T00:00:00Z","timestamp":1768780800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"KAKETSUKEN"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/vetsci13010098","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T13:02:07Z","timestamp":1768914127000},"page":"98","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Complete Neutralization of Tetanus Neurotoxin by Alpaca-Derived Trivalent Nanobodies Aimed at Veterinary Medical Applications"],"prefix":"10.3390","volume":"13","author":[{"given":"Chiyomi","family":"Sakamoto","sequence":"first","affiliation":[{"name":"Toxin and Biologicals Research Laboratory, Kumamoto Health Science University, 325 Izumi-machi, Kita-ku, Kumamoto 861-5533, Japan"},{"name":"The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), 8-7 Shinshigai, Chuou-ku, Kumamoto 860-0803, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chie","family":"Shitada","sequence":"additional","affiliation":[{"name":"Toxin and Biologicals Research Laboratory, Kumamoto Health Science University, 325 Izumi-machi, Kita-ku, Kumamoto 861-5533, Japan"},{"name":"The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), 8-7 Shinshigai, Chuou-ku, Kumamoto 860-0803, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norihiko","family":"Kiyose","sequence":"additional","affiliation":[{"name":"ARK Resource Co., Ltd., 383-2 Nakaharamachi, Nishi-ku, Kumamoto 861-5271, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuo","family":"Miyazaki","sequence":"additional","affiliation":[{"name":"ARK Resource Co., Ltd., 383-2 Nakaharamachi, Nishi-ku, Kumamoto 861-5271, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sena","family":"Kamesawa","sequence":"additional","affiliation":[{"name":"Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Morioka","sequence":"additional","affiliation":[{"name":"Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazunori","family":"Morokuma","sequence":"additional","affiliation":[{"name":"KM Biologics Co., Ltd., 1-6-1 Okubo, Kita-ku, Kumamoto 860-8568, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiko","family":"Tomokiyo","sequence":"additional","affiliation":[{"name":"The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), 8-7 Shinshigai, Chuou-ku, Kumamoto 860-0803, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-8404-4913","authenticated-orcid":false,"given":"Motohide","family":"Takahashi","sequence":"additional","affiliation":[{"name":"Toxin and Biologicals Research Laboratory, Kumamoto Health Science University, 325 Izumi-machi, Kita-ku, Kumamoto 861-5533, Japan"},{"name":"The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), 8-7 Shinshigai, Chuou-ku, Kumamoto 860-0803, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1111\/j.1939-1676.1994.tb03210.x","article-title":"Tetanus in the horse: A review of 20 cases (1970 to 1990)","volume":"8","author":"Green","year":"1994","journal-title":"J. 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