Functional and structural basis of Omicron BA.3.2.1 spike

SARS-CoV-2 BA.3.2 sublineages, derived from BA.3 and carrying substantial spike divergence, raised concerns about altered fitness and antigenicity. Using BA.3.2.1 as a representative strain, we engineered live-attenuated SARS-CoV-2 encoding BA.3.2.1, LP.8.1, or XEC spikes and benchmarked them against BA.3 and KP.3. BA.3.2.1 outcompetes BA.3 in primary human airway epithelium but replicates less efficiently than JN.1 descendants and shows the greatest resistance to neutralization by KP.2/KP.3 convalescent sera. Although BA.3.2.1 RBD binds hACE2 with high affinity, its trimeric spike engages hACE2 less efficiently than LP.8.1. Cryoelectron microscopy structures reveal that BA.3.2.1 spike predominantly adopts a compact, asymmetric closed conformation stabilized by protomer rearrangements, N-linked glycosylation, and a distinct fusion-peptide-proximal region. This architecture increases spike stability, limits receptor engagement, reduces fusogenicity, and masks antibody-sensitive epitopes. Thus, BA.3.2.1 enhances immune evasion at the cost of replication fitness, providing a structural-functional explanation for BA.3.2's limited prevalence and underscoring the need for continued variant surveillance.