Maturation-deficient chikungunya virus elicits protective immune responses in a murine challenge model

Maturation of chikungunya virus (CHIKV) particles is dependent on cleavage of the precursor glycoprotein p62 into the proteins E3 and E2 by the cellular protease furin. Here, we produced immature CHIKV particles by infecting furin-deficient cells and characterized them in comparison to mature particles by electron microscopy. Ectopic expression of furin in the furin-deficient cells restored the production of infectious particles, underpinning the importance of this enzyme in particle maturation. To prevent furin‑mediated maturation in mammalian cells, we engineered a recombinant CHIKV in which the furin cleavage site was replaced by a Tobacco Etch virus (TEV) protease site, rendering the virus non‑infectious unless treated in vitro with TEV protease. TEV‑matured particles became infectious and, when used for immunization, induced a stronger immune response than their untreated counterparts, as evidenced by higher neutralizing antibody titers. In vivo, a single immunization with TEV‑treated particles protected IFNAR‑/‑ mice against a lethal CHIKV challenge. In immunocompetent mice, vaccination also reduced viremia and CHIKV‑induced footpad swelling. Together, these findings demonstrate that in vitro maturation of CHIKV modified with a TEV cleavage site promotes a safe, replication limited immunogenic particle, establishing a promising platform for developing vaccines against viruses that require furin protease maturation.