Analysis of spike protein variants evolved in a novel mouse model of persistent SARS-CoV-2 infection

SARS-CoV-2 mutation rates have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants; however, there is currently no animal model to recapitulate persistent infection. We established a novel model of persistent infection using xenografts of Calu-3 human lung cancer cells in immunocompromised mice. After infection with wild-type SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three isolated viral clones with defined mutations produced higher virus titers than the wild-type virus in Calu-3 cells without cytotoxic effects. In K18-hACE2 mice, the variants were less lethal than the wild-type virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of wild-type S protein and provided protective immunity against subsequent challenge with wild-type virus. These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study persistent SARS-CoV-2 infection.