Ex vivo-stimulated cytokine expression in whole blood indicates swine influenza vaccine-induced immune memory.

Swine influenza viruses (SwIAV) pose a zoonotic threat and contribute to interspecies viral reassortment. Effective vaccination strategies are essential, yet accessible correlates of protection remain limited. We simplified a whole blood (WB) assay to assess immune memory ex vivo in pigs vaccinated with Respiporc FLU3® (n = 6) versus placebo controls (n = 6), followed by homologous challenge with SwIAV (H1avN1). Vaccinated pigs exhibited no lung lesions and minimal viral loads. WB samples, collected pre- and post-vaccination and at day 1 and 5 post-challenge (dpch), were stimulated with SwIAV to quantify IL2, IL4, IFNγ, TNFα, and granzyme B (GZMB) mRNA via qPCR. High IL2 and moderate IL4 mRNA elicited in WB post-booster predicted reduced viral load and lung pathology. Concomitantly, high IFNγ in WB from controls at 5dpch correlated with lesion severity, indicating excessive inflammation in non-vaccinates. Post-challenge, IL4/IL2 mRNA ratios increased in virus-stimulated WB of vaccinates, suggesting Th2-associated resolution of infection. In contrast, controls showed declining IL4/IL2 ratios post-challenge, correlating with disease severity. Thus, the simplified WB assay allows identification of virus-specific immune responses associating with protection, without requiring extensive laboratory infrastructure. By recording the ex vivo-triggered SwIAV-specific IL4/IL2 mRNA dynamics, which develop between booster and challenge monitoring immune memory may simplify determination of vaccination success.