Reoviridae-derived short trimerization domain for stabilizing homo-trimerization of vaccine immunogens and therapeutic proteins

Trimerization motifs play pivotal roles in structural biology and therapeutic protein engineering. Here, we engineered a novel short trimerization motif (rFd1303) derived from the Reoviridae family reovirus σ1 protein. Compared with the widely used T4-Foldon, the rFd1303 enhanced thermal stability and increased recombinant protein yields. The rFd1303-fused immunogens of SARS-CoV-2 spike and influenza hemagglutinin elicited antibody responses comparable to T4-Foldon-fused controls in murine models. Leveraging the new tag, we engineered the trimeric ACE2-Ig (TriACE2-Ig), which exhibits exceptional stability (room-temperature storage for 30 days) and broad neutralization against multiple SARS-CoV-2 variants (average IC ₅₀ of 2.9 ng/mL), showing a 20.9-fold potency improvement over monomeric ACE2-Ig. In the hamster model challenged with various SARS-CoV-2 variants, our data demonstrated that intranasal TriACE2-Ig administration markedly reduced viral loads, virus-induced body-weight loss, lung pathology, and decreased within-cage virus transmission. These findings highlight rFd1303 as a versatile trimerization platform for vaccine and therapeutic protein development.