Altered infectivity, cell-cell fusion, and immune evasion of SARS-CoV-2 BA.3.2 and LP.8.1 variants.

The continued evolution of SARS-CoV-2 has yielded highly immune-evasive Omicron subvariants that challenge existing population immunity. In late 2024, a novel subvariant designated BA.3.2 emerged independently from the BA.3 lineage, carrying over 50 spike mutations, and representing a distinct evolutionary branch with unclear biological properties. Here, we characterize the BA.3.2 spike-mediated entry, fusogenicity, antigenicity, and neutralization in comparison to its parental BA.3- and JN.1-derived co-circulating LP.8.1 subvariants. Using lentiviral pseudotyping assays, we show that BA.3.2 exhibits significantly reduced infectivity in both 293T-ACE2 and CaLu-3 cells relative to BA.3, accompanied by attenuated spike-mediated cell-cell fusion. Despite its impaired entry, BA.3.2 evades neutralizing antibodies (nAbs) elicited by bivalent mRNA vaccination, Omicron BA.1-wave, and JN.1-wave infection, with a > 25-fold reduction in nAb titers compared to BA.3 in both bivalent mRNA vaccination and Omicron BA.1-wave cohorts. Antigenic mapping confirms remarkable divergences of BA.3.2 and LP.8.1 from the ancestral D614G, Omicron BA.3, and JN.1. Mechanistically, BA.3.2 acquires four new N-linked glycosylation sites in the N-terminal domain (NTD) and receptor-binding domain (RBD) of spike; disruption of which enhances viral infectivity and restores sensitivity to nAbs, suggesting a functional trade-off between immune evasion and spike functionality. Together, our findings define BA.3.2 as a highly immune-evasive variant with altered spike properties, shaped in part by glycan alteration. These results underscore the ongoing antigenic diversification of SARS-CoV-2 and highlight the importance of monitoring independent evolutionary trajectories outside the dominant JN.1 lineage. IMPORTANCE: The Omicron subvariant BA.3.2 has independently evolved from an early BA.3 lineage, carrying over 50 amino acid substitutions in its spike protein. Our study demonstrates that BA.3.2 exhibits markedly reduced infectivity and fusion activity but strong resistance to neutralizing antibodies elicited by vaccination or prior Omicron infection. We further show that newly acquired N-linked glycans in both the N-terminal and receptor-binding domains of BA.3.2 contribute to immune escape, while impairing spike-mediated entry. These findings reveal that glycan remodeling represents a key mechanism driving SARS-CoV-2 antigenic diversification and functional trade-offs between immune evasion and infectivity. Monitoring such independently evolving Omicron lineages is essential for understanding ongoing viral adaptation and for guiding future vaccine design.