Structural mechanisms behind the neutralisation of long-chain α-neurotoxins by broadly neutralising VHHs discovered using a consensus antigen

Snakebite envenoming, a neglected tropical disease, affects millions globally, causing significant morbidity and mortality. Developing broadly neutralising monoclonal antibodies offers a promising approach to address the antigenic variation present in snake venoms. In this study, we designed a long-chain consensus α-neurotoxin, LCC, to serve as an antigen in a phage display–based antibody discovery campaign. Utilising a yeast expression system, we expressed LCC and identified 21 variable domains of heavy-chain-only antibodies (VHHs) from immune libraries. These VHHs were assessed for their binding affinity to various long-chain α-neurotoxins and their neutralising capability in vitro. The VHH with the broadest cross-reactivity and highest affinity, TPL1158_01_C09, was co-crystallised with α-cobratoxin to elucidate its binding mechanism. In vivo rodent studies demonstrated the neutralisation potential of TPL1158_01_C09. Our findings highlight that the use of a consensus toxin as an antigen coincided with the discovery of broadly neutralising VHHs against snake venom toxins.

Keywords: Q3 2025