Xenoglycosylation of the HIV-1 Glycan Shield Promotes Cross-Clade Neutralization and Diverse Human Antibody Lineages

Despite advances in HIV-1 envelope glycoprotein (Env)-based vaccine design, eliciting broadly neutralizing antibodies (bNAbs) remains a central challenge. Here, we identify core α(1,3)-fucose as a key modulator of neutralizing antibody (NAb) induction and breadth. Uncleaved prefusion-optimized (UFO) trimers and self-assembling protein nanoparticles (SApNPs) produced in High Five (Hi5) insect cells incorporate core α(1,3)-fucose into the Env glycan shield. Crystal structures of Hi5-produced BG505 UFO trimers in complex with bNAbs confirm native-like Env architecture and epitope presentation. In wild-type mice, these trimers and SApNPs elicit robust autologous and heterologous NAb responses. We then engineer Chinese hamster ovary (CHO) cells to co-express three insect- and plant-derived core α(1,3)-fucosyltransferases. The resulting immunogens carry fucosylated mammalian glycans and elicit cross-clade NAb responses in mice, supporting an important role for core α(1,3)-fucose in broadened neutralization. Env-specific IgE responses suggest engagement of associated immune pathways during vaccination. In ATX-Gx humanized mice, core α(1,3)-fucosylated, glycan-trimmed Env elicits detectable cross-clade neutralization by week 11. Single-cell analysis identifies distinct human antibody lineages with cross-clade activity and noncanonical Env recognition. Repertoire and functional analyses uncover putatively related lineage members across two animals within the same group. Together, these findings support xenoglycosylation as a promising, germline-agnostic strategy for HIV-1 vaccine design.