Integrated Systems Vaccinology Reveals Distinct Metabolic Responses to SARS-CoV-2 Infection and DNA-Based Vaccines in Ferrets

Understanding systemic effects of vaccination and infection is central to defining correlates of protection against SARS-CoV-2. We used untargeted serum metabolomics to profile the immunometabolic landscape of ferrets after SARS-CoV-2 infection and DNA-/protein-based vaccination. Ferrets were vaccinated with either a multigenic DNA vaccine encoding SARS-CoV-2 RBD, M, and N (OC2), an N-only DNA vaccine (OC12), a recombinant spike protein with QS-21 adjuvant (S+QS21), or a hepatitis B/D control construct (Hep-B/D), and subsequently challenged with SARS-CoV-2. Serum was analyzed longitudinally at baseline, post-vaccination, and post-challenge. SARS-CoV-2 infection induced broad metabolic reprogramming, involving TCA cycle, glutathione metabolism, and nucleotide turnover, reflecting inflammation and cellular activation. OC2 vaccination induced strong metabolic shifts in amino acid and mitochondrial pathways despite low pre-challenge anti-S antibodies. Post-challenge, these shifts extended to redox and nucleotide pathways, correlating with robust anti-S and very strong anti-N antibody responses and complete viral clearance in BAL, but with marked airway pathology, consistent with T cell-mediated clearance of infected cells. S+QS21 and OC12 induced distinct, immunogen-specific signatures with partial protection, while Hep-B/D showed minimal systemic engagement. Metabolite-antibody correlations revealed vaccine-specific associations, highlighting lipid and amino acid pathways as potential immunogenicity biomarkers. Overlap and heatmap analyses showed that metabolic trajectories reflect both the magnitude and quality of immune training. These findings underscore the value of systems vaccinology in resolving mechanistic differences in vaccine responses and support metabolic profiling as a tool for evaluating immune efficacy in preclinical vaccine studies.