Top-down mass spectrometry reveals O-glycoform structural changes in the SARS-CoV-2 Omicron variant. Resolving the mutations and post-translational alterations can inform strategies for designing variant-directed diagnostics and therapeutics.

The SARS-CoV-2 Omicron (B.1.1.529) variant possesses numerous spike (S) mutations particularly in the S receptor-binding domain (S-RBD) that significantly improve transmissibility and evasion of neutralizing antibodies. But exactly how the mutations in the Omicron variant enhance viral escape from immunological protection remains to be understood. The S-RBD remains the principal target for neutralizing antibodies and therapeutics, thus new structural insights into the Omicron S-RBD and characterization of the post-translational glycosylation changes can inform rational design of vaccines and therapeutics. Here we report the molecular variations and O-glycoform changes of the Omicron S-RBD variant as compared to wild-type (WA1/2020) and Delta (B.1.617.2) variants using high-resolution top-down mass spectrometry (MS). A novel O-glycosite (Thr376) unique to the Omicron variant is identified. Moreover, we have directly quantified the Core 1 and Core 2 O-glycan structures and characterized the O-glycoform structural heterogeneity of the three variants. Our findings reveal high resolution detail of Omicron O-glycoforms and their utilization to provide direct molecular evidence of proteoform alterations in the Omicron variant which could shed light on how this variant escapes immunological protection.

Influenza remains a major global health concern, with the ongoing seasonal epidemics causing millions of cases and up to 650,000 deaths worldwide annually. Current influenza vaccines only provide strain-specific and short-lived protection, exposing vulnerability to antigenic drift and reassortments. To overcome these limitations, next-generation vaccine platforms are being developed, with nanoparticle-based approaches showing promise. Displaying hemagglutinin (HA) on multivalent scaffolds enhances B cell receptor engagement, germinal center formation, and affinity maturation, while supporting durable and broadly protective humoral immunity. This review highlights recent published advances found in PubMed, Web of Science, and Google Scholar since 2020 in HA-displaying nanoparticle influenza vaccines, emphasizing strategies to improve immunogenicity, broaden protection across influenza strains and subtypes, and redirect responses toward conserved epitopes of HA.

Influenza has been a global health concern for the past century. Current seasonal influenza vaccines primarily elicit an antibody response that targets the immunodominant head domain of the viral glycoprotein hemagglutinin (HA), which consistently mutates due to selective pressure. To circumvent this problem, we introduce a “tethered antigenic suppression” strategy to shield the HA head domain and refocus the immune response towards the conserved but immunosubdominant stalk domain of HA. Specifically, we tethered an antibody fragment (Fab) that recognizes the Sb antigenic site in the HA head domain to the HA protein with a linker. We immunized separate groups of female mice with the Fab-tethered HA or regular HA and characterized the elicited antibody response. We demonstrate that shielding the HA head domain with a tethered Fab suppresses the antibody titers towards all five key antigenic sites in the HA head domain while eliciting a robust anti-stalk antibody response. Our work highlights the potential of tethered antigenic suppression as a strategy to refocus the antibody response towards conserved epitopes on protein antigens.

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present tremendous challenges to study using conventional structural biology techniques. Here we develop a “native nanoproteomics” strategy for the native enrichment and subsequent native top-down mass spectrometry (nTDMS) of of low-abundance protein complexes. Specifically, we demonstrate the first comprehensive characterization of the structure and dynamics of cardiac troponin (cTn) complexes directly from human heart tissue. The endogenous cTn complex is effectively enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complexes, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the heterotrimeric cTn complex, localizes Ca ²⁺ binding domains (II-IV), defines cTn-Ca ²⁺ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a new paradigm for structural characterization of low-abundance native protein complexes.