Yellow fever vaccine propagation in primary human hepatocytes triggers antiviral and cytolytic responses

Despite the long-standing success of the yellow fever virus (YFV) 17D vaccine, the precise virus and host determinants which confer its virulence attenuation remain incompletely defined. To provide insights, we performed YFV genome-level evolutionary analyses combined with investigations of vaccine hepatotropism. Evolutionary analyses confirmed vaccine-specific mutations are selected against in virulent YFV isolates, with genomic signatures of purifying selection most pronounced in the envelope (E) protein, where vaccine-specific mutations are concentrated. Structural prediction and mapping using Alphafold3 confirmed vaccine-specific mutations form a surface-exposed patch on domain III of the E protein, likely overlapping the flavivirus receptor binding domain. In tissue culture, 17D infections exhibited enhanced cell-to-cell spread and cytolysis compared to virulent YFV strains. In humans, YFV infections cause severe liver damage while vaccination has an excellent safety profile. Consequently, we next investigated whether 17D displays impaired tropism for primary human hepatocytes (PHH) or induces distinct cell-intrinsic responses to virulent strains. Surprisingly, ex vivo infections confirmed robust propagation of both 17D and virulent YFV strains in PHH. RNA-sequencing revealed consistent and shared induction of IFNB and IFNL1-4, modulating gene programs associated with antiviral responses, cellular immunity, chemotaxis and inflammation, cell-death, metabolism and protein translation. Consistent with differences in spreading efficiency, subtle differences in virion production kinetics and the magnitude and tempo of PHH transcriptional responses were observed. Antiviral responses to 17D were activated earlier while responses to virulent YFV were delayed but enhanced. In summary, we confirm the YFV vaccine exhibits comparable hepatotropism to virulent strains and induces virtually identical host responses. Mechanistically, these data suggest that impaired hepatotropism and associated cytopathology are not correlates of 17D’s virulence attenuation, implying unknown barriers to systemic dissemination mitigate viscerotropic spread and associated liver pathology upon vaccination.