Evanson, L. R. | Search bioRxiv

bioRxiv2026

Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry

While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line-dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air-liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line-derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.

microbiology↗

bioRxiv2025

Unconventional linkers facilitate potent stabilized coronavirus stem antibody responses following nanoparticle vaccination

Vaccine technologies that protect against a range of related pathogens within viral families, such as human immunodeficiency virus (HIV), influenza, and coronaviruses (CoVs) represent the future of viral vaccine development. Towards developing broad-spectrum CoV and influenza vaccines, we and others previously designed and evaluated CoV and influenza stem antigens; but these elicited relatively weak and sub-neutralizing antibody (Ab) responses. Multivalent antigen display on nanoparticles (NPs) is an established strategy to enhance and shape immunogenicity. However, one facet of NP vaccines has been largely overlooked: the indispensable linker segment between the antigen and NP core. Here, we introduce de novo-designed rigid (L2) and rarely used long flexible (L6) linkers to optimally display antigens on NPs, target occluded epitopes, and enhance cross-reactive Ab responses, using prefusion-stabilized Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S-2P) and stem (SS) antigens as prototype antigens. Antigenic characterization of L2-NPs confirmed enhanced Ab binding and exposure of cross-reactive epitopes compared with L6-NPs and soluble antigens. Immunization with SS-L2-NPs elicited broader, more potent cross-reactive Ab responses across the seven human-infecting CoVs and pandemic threat WIV1-CoV, whereas SS-L6-NPs induced stronger neutralizing Ab responses against MERS-CoV, SARS-CoV-2, and WIV1-CoV. Ab competition and systems serology analyses revealed that SS-L2-NPs elicit robust Fc-mediated effector functions. By improving CoV-targeting Ab functionality, these linker approaches have the potential to confer broad-spectrum CoV protection and represent a promising strategy against hypervariable influenza and HIV viruses - as well as other broad viral families with pandemic potential.

bioengineering↗