Characterization of Immune Responses to RBD-SD1 Nanoparticle Vaccines Derived from ACE2-Capable HKU5-CoV-2, NeoCoV, and HKU5 Merbecoviruses.

Recent discoveries of merbecoviruses such as HKU5, NeoCoV, and HKU5-CoV-2 that use angiotensin-converting enzyme 2 (ACE2) as their entry receptor underscore the potential for future spillover to humans. To combat these ACE2-utilizing merbecoviruses, we developed virus-like particle (VLP) vaccines displaying their receptor-binding domain and subdomain 1 (RBD-SD1) antigens fused to SpyTags. These antigens were then conjugated to SpyCatcher-mi3 nanoparticles. Immunization of mice with each RBD-SD1-mi3 vaccine produced robust IgG responses against homologous antigens and measurable but reduced binding to heterologous RBD-SD1 proteins. Antigenic cartography showed that antigenic relationships broadly reflected sequence identity with HKU5 positioned between NeoCoV and HKU5-CoV-2. Despite HKU5 and HKU5-CoV-2 sharing the greatest genetic similarity, cross-reactive titers between these two groups were not substantially higher than those observed with NeoCoV. In a surrogate virus neutralization test (sVNT), sera from mice vaccinated with NeoCoV RBD-SD1-mi3 or HKU5-CoV-2 RBD-SD1-mi3 showed strong inhibition of receptor binding by their respective Fc-fused RBD-SD1 proteins. Sera from mice vaccinated with HKU5 RBD-SD1-mi3 exhibited some cross-inhibition activity against both NeoCoV and HKU5-CoV-2 Fc-fused RBD-SD1. Together, these findings show that the RBD-SD1-displaying nanoparticle vaccine can elicit cross-reactive antibodies against ACE2-binding merbecoviruses and that antigenic relationships are shaped by more than sequence similarity.