Spatial transcriptomic mapping of early immune responses to an adjuvanted recombinant hemagglutinin vaccine

Vaccination relies on innate and adaptive responses initiated at the injection site followed by activation in draining lymph nodes. However, high-plex, spatially resolved maps that couple injection site signals with transcriptomic programs in draining lymph nodes over time are still lacking. Here, we used spatial transcriptomics to map early responses to an adjuvanted recombinant haemagglutinin (rHA) protein vaccine formulated with Sanofi's proprietary oil-in-water adjuvant AF03. Mice received intramuscular rHA-protein or PBS. Injection site muscle and draining lymph nodes were collected at Days 1 and 7 post-prime for GeoMx™ Digital Spatial Profiling (DSP) and CosMx™ Spatial Molecular Imaging (SMI) single-cell imaging. In lymph nodes, rHA-protein vaccination induced early innate pathway activation in non-follicular regions at Day 1. By Day 7, adaptive pathways were enriched within follicles, with differentially expressed genes (DEGs) linked to germinal center (GC) formation, transcriptional regulation and signaling. These changes coincided with expansion of a proliferative GC-like B-cell cluster within the follicular area. At the injection site, 20x20 µM spatial grids were used rather than segmented cells. Clusters, enriched for fibroblast and muscle cells showed Day 1 induction of chemokines and interferon stimulated genes, concentrated in fibroblast rich stromal layers. These spatially organized tissue programs were accompanied by transient systemic cytokine elevations and increased antigen-specific IgG titers. Together, these data provide a previously unresolved view of what, when and where local inflammation and lymph node organization are spatially and temporally associated during early responses following vaccination with an adjuvanted rHA-protein vaccine.