Characterization of Immune Responses to RBD‐SD1 Nanoparticle Vaccines Derived From ACE2‐Capable HKU5‐CoV‐2, NeoCoV, and HKU5 Merbecoviruses

ABSTRACT

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.