Antigenic divergence of cobra short-chain α-neurotoxins: Implications for regional antivenom effectiveness in Southeast Asia

Short-chain α-neurotoxins (SNTXs) are the principal neurotoxic components in several Asiatic cobras, including Naja atra, Naja philippinensis, and Naja samarensis. Although structurally conserved, SNTXs exhibit marked antigenic variation that can limit the effectiveness of regional antivenoms used for snakebite envenoming in Asia. Here, we evaluated the immunoreactivity of the Philippine Cobra Antivenom (PCAV) and three regional products-Naja kaouthia Monovalent Antivenom (NkMAV), Neuro Bivalent Antivenom (NBAV), and Indonesian SABU-against a panel of cobra venoms and purified α-neurotoxins. PCAV bound strongly to homologous N. philippinensis SNTX but showed significantly lower cross-reactivity with SNTXs from Naja kaouthia, Naja sputatrix, and N. atra, and minimal recognition of marine elapid SNTXs and long-chain α-neurotoxins (LNTXs) of Monocled Cobra as well as King Cobra. Conversely, NkMAV and NBAV recognized mainland Asian SNTXs more broadly but reacted poorly with the Philippine cobra toxin. These differences were statistically significant in multiple comparisons versus the homologous venom control. Hierarchical clustering of normalized immunoreactivity delineated two major SNTX antigenic subtypes corresponding to Philippine versus continental Asian lineages. Peptide sequence analysis unmasked two distinct loop-II motifs (28WWS-TII37 and 28RWR-YRT37) associated with these divergent immunotypes. Phylogenetic reconstruction suggests that the Philippine cobras retain an ancestral-like SNTX motif, while Sundaic and East Asian cobras have diversified to employ another major SNTX form. Epitope prediction further revealed differences in surface exposure and accessibility, which helps explain the limited cross-neutralization among antivenoms. These findings demonstrate a clear antigenic dichotomy among Asian cobra SNTXs, which underlies species-specific antivenom effectiveness and highlights the need to incorporate representative SNTX variants into immunogen formulations to improve regional antivenom coverage.