Heat-Killed Enterococcus faecalis EF-2001 Promotes Systemic Th1-Skewed Immune Activation Without Detectable Reduction of Influenza Viral Load in Mice

Heat-killed <i>Enterococcus faecalis</i> EF-2001 (EF-2001) is a postbiotic preparation reported to modulate host immunity. However, its specific impact on host immune responses and virological outcomes during the early phase of influenza infection remains insufficiently characterized. Female BALB/c mice received oral EF-2001 (16 mg/kg/day) for either 4 days or 14 days prior to intranasal inoculation with influenza A/H3N2 (A/Aichi/2/68). On day 2 post-infection, splenic T-cell subsets (CD3⁺, CD4⁺, CD8⁺) were quantified by flow cytometry. Cytokines released from PMA/ionomycin-stimulated splenocytes were measured using a cytometric bead array assay to assess functional polarization. Lung viral titers (TCID₅₀) and interferon-α (IFN-α) concentrations were assessed to evaluate local antiviral efficacy. EF-2001 administration significantly increased the proportions of splenic CD3⁺ T cells, including both CD4⁺ and CD8⁺ subsets, compared to controls. The 14-day pretreatment regimen significantly enhanced IFN-γ production while reducing IL-10, IL-4, and IL-2 secretion, consistent with a distinct systemic Th1-skewed immune activation. In contrast to these systemic effects, EF-2001 did not significantly reduce lung viral titers (difference < 0.2 log₁₀ TCID₅₀) and did not increase lung IFN-α concentrations at day 2 post-infection. Oral EF-2001 pretreatment promoted systemic immune activation characterized by T-cell expansion and a Th1-biased cytokine profile. However, this systemic priming showed no detectable antiviral effect on lung viral burden at the early evaluation time point. EF-2001 may be better positioned as an adjunctive immunomodulatory approach rather than a direct antiviral agent, warranting further studies that include clinical outcomes and multi-time-point antiviral and mucosal immune assessments.