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 describe a short trimerization motif (rFd1303) derived from the Reoviridae family reovirus σ1 protein. Compared with the widely used T4-Foldon, the rFd1303 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 mice. Using this tag, we engineered a trimeric ACE2-Ig fusion protein (TriACE2-Ig) that remained stable after 30 days at room temperature and neutralized a broad range of ACE2-utilizing sarbecoviruses, including 19 SARS-CoV-2 variants, SARS-CoV, and pangolin coronaviruses GD and GX, with higher potency than 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. In this study, the authors engineer a short trimerization tag for vaccine and therapeutic protein development. The tag enables a trimeric ACE2 decoy that exhibits broad neutralizing activity against coronaviruses and confers protection in animal models.