A multimodal in situ framework for spatial characterization applied on SARS-CoV-2 live-attenuated vaccines in the nasal mucosa.

Respiratory viruses frequently initiate infection in the nasal mucosa, where virus-host interactions shape disease progression and transmission. Yet this site remains undercharacterized due to anatomical and technical challenges. Spatially resolved technologies are limited by cost and complexity, leaving most studies reliant on bulk or descriptive approaches lacking spatial context. We present a framework for spatially resolved investigation of infections in the nasal cavity. Using wild-type SARS-CoV-2 and two live-attenuated vaccines (OTS-206, OTS-228) as model viruses in Syrian hamsters, we integrate standardized sampling, histopathology, immunohistochemistry, multiplex RNA in situ hybridization, and quantitative image analysis across spatial scales, including tissue, single-cell, and subcellular resolution. We identified distinct infection patterns and graduated tissue damage from wild-type to OTS-228. Despite limited infection, OTS-228 induces a robust tissue-preserving immune response. Subcellular analysis shows that both vaccines exhibit a primarily cytoplasmic distribution of host antiviral transcripts, whereas WT infection is characterized by predominantly nuclear RNA localization.