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

TL;DR

A heat-killed bacteria called EF-2001 boosts the immune system in mice but does not reduce influenza virus levels in the lungs.

Contribution

EF-2001 induces systemic Th1-skewed immune activation without reducing early influenza viral load in mice.

Findings

EF-2001 increased CD3+, CD4+, and CD8+ T-cell proportions in the spleen.

The 14-day regimen enhanced IFN-γ and reduced IL-10, IL-4, and IL-2, indicating Th1 polarization.

EF-2001 had no significant effect on lung viral titers or IFN-α levels at day 2 post-infection.

Abstract

Heat-killed Enterococcus faecalis 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.

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1. Introduction

The intestinal microbiota is now recognized as a critical regulator of host physiology and immune homeostasis. Through the production of microbial-associated molecular patterns (MAMPs)—such as peptidoglycan, lipopolysaccharides, and flagellin—and the synthesis of bioactive metabolites like short-chain fatty acids (SCFAs), gut bacteria shape both local mucosal immunity and systemic immune responses. This distant regulation is conceptually framed as the “gut-lung axis,” a bidirectional communication network whereby gut-derived immune signals influence susceptibility to and outcomes of respiratory infections, including influenza viruses and coronaviruses [1,2]. Mechanisms underlying this axis include the migration of immune cells imprinted in the gut-associated lymphoid tissue (GALT) to the respiratory tract via CCR9/CCR10 signaling pathways, and the systemic circulation of metabolites that enhance the microbicidal activity of alveolar macrophages [2,3].

Historically, immunomodulation strategies have focused on probiotics (live microorganisms) and prebiotics (substrates for beneficial microbes). However, the use of live probiotics presents challenges, including strict cold-chain requirements for viability and theoretical safety concerns—such as bacteremia or transfer of antibiotic resistance genes—particularly in immunocompromised or critically ill populations [4]. Consequently, scientific interest has expanded to “postbiotics,” defined by the International Scientific Association for Probiotics and Prebiotics (ISAPP) as “preparations of inanimate microorganisms and/or their components that confer a health benefit on the host [4].” Heat-killed bacterial preparations represent a major class of postbiotics, offering significant practical advantages such as extended shelf-life, standardized dosing, and enhanced safety profiles while retaining immunogenic cell wall components capable of engaging pattern-recognition receptors (PRRs) [4,5].

Enterococcus faecalis EF-2001 (EF-2001) is a commercially available heat-killed postbiotic preparation derived from a specific human isolate. Previous preclinical studies have demonstrated its biologic biological activity in diverse contexts. For instance, EF-2001 has been reported to ameliorate dinitrobenzene sulfonic acid (DNBS)-induced inflammatory bowel disease in mice by downregulating pro-inflammatory cytokines [1], and to exhibit anti-tumor and anti-allergic effects in other models [6]. Reviews of Enterococcus-derived preparations suggest they possess broad immunoregulatory potential, likely mediated through the interaction of cell wall lipoteichoic acid (LTA) and peptidoglycan with Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain (NOD) proteins [7,8].

Despite these promising immunomodulatory properties, the impact of heat-killed EF-2001 on host defenses against acute respiratory viral infections remains insufficiently characterized. Influenza A virus infection continues to be a major global health burden, inducing a complex immune response where the balance between viral clearance and immunopathology is critical [9]. Effective clearance of intracellular pathogens like influenza typically requires a Type 1 helper T cell (Th1) response, characterized by the production of interferon-gamma (IFN-γ) and the activation of cytotoxic T lymphocytes (CTLs). Conversely, an excessive Type 2 (Th2) response or regulatory T cell (Treg) suppression during the early phase may delay viral clearance or alter disease severity [10].

Several live probiotic strains, including species of Lactobacillus and Bifidobacterium, have shown influenza-protective effects in animal models, often associated with enhanced Type I interferon responses, alveolar macrophage activation, and Th1-type immunity [11,12,13,14,15,16]. However, it is unclear whether non-viable, heat-killed EF-2001 can elicit similar protective systemic or mucosal alterations. In this study, we examined whether oral pretreatment with heat-killed EF-2001 alters systemic immune profiles (T-cell subsets and cytokine polarization) and/or virological outcomes in a murine influenza A infection model. We hypothesized that EF-2001, acting as a TLR agonist via the gut mucosa, would modulate systemic T-cell composition and enhance Th1-type cytokine responses, although its ability to suppress lung viral replication in the absence of live bacterial metabolites remained uncertain.

2. Materials and Methods

2.1. EF-2001 Preparation

Heat-killed Enterococcus faecalis EF-2001 (Lot No. 230315-1) was provided as a commercially standardized dried powder by Nihon BRM Co. (Tokyo, Japan). The preparation consists of bacteria that have been heat-treated to ensure non-viability while preserving cell wall structural integrity. According to manufacturer specifications, one gram of the powder corresponds to approximately 7.5 × 10^16^ colony-forming unit (CFU) equivalents prior to inactivation. The powder was stored at 4 °C in a desiccated environment until use. For oral administration, EF-2001 was suspended in sterile Dulbecco’s phosphate-buffered saline (PBS) immediately before daily gavage to achieve the target dosage.

2.2. Animals and Ethics

Six-week-old female BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). Mice were housed in a specific-pathogen-free (SPF) facility at Aichi Medical University under controlled conditions: temperature 23 ± 2 °C, humidity 50 ± 10%, and a strict 12-h light/12-h dark cycle. Animals had ad libitum access to standard irradiated rodent chow and sterilized water. Mice were allowed to acclimatize for one week prior to the initiation of experiments. Mice were rendered unconscious under inhalational sevoflurane anesthesia, and euthanized by overdose administration of pentobarbital. All experimental procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Aichi Medical University (Approval No. 2024-64). Also, this animal experiments were carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 (https://arriveguidelines.org accessed on 24 January 2026). Animal experimental procedures including euthanasia methods were performed according to the SCIENCE COUNCIL OF JAPAN (SCJ)’s publication guide for care and use of laboratory animals (https://www.scj.go.jp/ja/info/kohyo/pdf/kohyo-20-k16-2.pdf accessed on 24 January 2026), and associated guidelines. To minimize suffering, humane endpoints were established, although no animals reached these endpoints prior to the scheduled sacrifice.

2.3. Study Design and Influenza Infection

To evaluate the effect of administration duration, mice (n = 15) were randomly assigned to three experimental groups (n = 5 per group) using a block randomization method:

1. Control (PBS): Received oral vehicle (0.1 mL sterile PBS) once daily via gavage on days −4 to −1 relative to infection.
2. EF-2001 (4-day): Received EF-2001 at 16 mg/kg/day (equivalent to 2.4 × 10^15^ CFU equivalents/mouse/day) on days −4 to −1.
3. EF-2001 (14-day): Received EF-2001 at 16 mg/kg/day on days −14 to −1 to assess the effects of longer-term priming.

The dosage was selected based on previous efficacy studies in murine colitis models [1]. On day 0, mice were anesthetized via intraperitoneal injection of a ketamine/xylazine mixture to ensure deep sedation and prevent sneezing reflex during inoculation. While anesthetized, mice were intranasally inoculated with influenza A/H3N2 (strain A/Aichi/2/68). Standardized procedures and techniques were used to control variability in anesthesia and intranasal inoculation techniques based on the previous guidance. The viral stock (1.1 × 10^13^ PFU/mL) was diluted in cold PBS to 1.1 × 10^11^ PFU/mL, and 50 μL was administered dropwise into the nares (total inoculum: 5.5 × 10^2^ PFU/mouse). This viral dose was calibrated to cause sublethal infection allowing for immune analysis without high mortality. Mice were monitored daily and euthanized on day +2 post-infection to evaluate early innate and adaptive immune activation. Spleens and lungs were aseptically harvested for analysis.

2.4. Flow Cytometry Analysis

Single-cell suspensions were prepared from samples, filtered through a 70-µm cell strainer, and washed in PBS containing 2% FBS. Cells were counted, and 1–2 × 10^6^ cells were used per staining. Cells were incubated with an Fc receptor–blocking reagent for 10 min at 4 °C, followed by staining with a fixable viability dye and fluorochrome-conjugated monoclonal antibodies in Table 1 for 20–30 min at 4 °C in the dark. For intracellular staining, cells were fixed and permeabilized, and stained with antibodies.

Data were acquired on a flow cytometer with single-stained compensation controls and fluorescence minus one (FMO) controls when indicated. At least 50,000–200,000 events were collected per sample. The gating strategy was as follows: (1) exclusion of debris via FSC-A vs. SSC-A; (2) singlet discrimination via FSC-A vs. FSC-H; (3) identification of live CD45^+^ leukocytes; (4) selection of CD3^+^ T cells; and (5) subdivision into CD4^+^ and CD8^+^ populations. Analysis was performed using FlowJo (v10.9.0, BD Biosciences) by an investigator blinded to the treatment group, and gating was applied sequentially to exclude debris (FSC/SSC), doublets (FSC-A vs. FSC-H), and dead cells, followed by identification of target populations based on marker expression.

2.5. Ex Vivo Stimulation and Cytokine Measurement

To assess the functional polarization of T cells, splenocytes were resuspended in complete RPMI-1640 medium containing 10% FBS and seeded at 1 × 10^12^ cells/mL in 24-well plates. Cells were stimulated non-specifically with Phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (1 μg/mL) for 48 h at 37 °C in 5% CO_2_. This long stimulation protocol is generally procedure to elicit maximal cytokine production capability. Supernatants were collected after centrifugation (600× g, 10 min, 4 °C) and stored at −80 °C.

Concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF were quantified using the BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit according to the manufacturer’s instructions (https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/CBA_MouseTh1Th2Th17_Kit_Manual.pdf accessed on 24 January 2026): the theoretical limit of detection for each cytokine are 0.1 pg/mL for IL-2, 0.03 pg/mL for IL-4, 1.4 pg/mL for IL-6, 16.8 pg/mL for IL-10, 0.8 pg/mL for IL-17A, 0.5 pg/mL for INF-γ and 0.9 pg/mL for TNF. Samples were acquired on the LSRFortessa X-20 (BD Biosciences, Tokyo, Japan), and data were analyzed using FCAP Array software version 3.0. The lower limit of detection was approximately 20 pg/mL for most analytes. In each group, IL-17A and TNF were consistently below the limit of detection. To ensure data robustness, they were excluded from the final analysis.

2.6. Lung Viral Titration (TCID50) and IFN-α ELISA

Lungs were weighed and homogenized in 1 mL of cold PBS. Homogenates were centrifuged at 7500× g for 5 min to pellet debris. For viral titration, supernatants were assayed on Madin-Darby Canine Kidney (MDCK) cells. MDCK monolayers (1.5 × 10^11^ cells/well) were prepared in 96-well plates 24 h prior. Ten-fold serial dilutions (10 to 10) of lung supernatant were adsorbed onto cells for 1 h, washed, and replaced with serum-free DMEM containing 2 μg/mL TPCK-treated trypsin. Plates were incubated for 72 h, and the cytopathic effect (CPE) was visually scored. The 50% Tissue Culture Infectious Dose (TCID_50_) was calculated using the Reed–Muench mathematical method. Lung IFN-α levels were determined from the same supernatants using the VeriKine Mouse IFN-α ELISA kit (PBL Assay Science, Tokyo, Japan), with absorbance read at 450 nm on a SpectraMax ABSPlus microplate reader (Molecular Devices, Tokyo, Japan).

2.7. Statistical Analysis

Sample size was determined based on previous experience with murine influenza models to detect a 1-log difference in viral titer with 80% power at alpha = 0.05. Statistical analyses were performed using R statistical software version 4.3.1. Data are presented as mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified using Levene’s test. Comparisons among the three groups were conducted using one-way Analysis of Variance (ANOVA). When the F-test was significant, post-hoc pairwise comparisons were performed using the Tukey–Kramer test to control for family-wise error rate. A two-sided p-value < 0.05 was considered statistically significant. Significance levels are denoted in figures as * p < 0.05 and ** p < 0.01.

3. Results

3.1. EF-2001 Administration Increased the Proportion of Splenic T-Cell Subsets Following Influenza Infection

To evaluate whether oral EF-2001 pretreatment alters the systemic cellular immune landscape during early influenza infection, we analyzed splenic lymphocyte populations on day 2 post-infection. Flow cytometric analysis revealed that EF-2001 administration significantly modulated T-cell abundance. Mice in the 14-day EF-2001 pretreatment group exhibited a statistically significant increase in the frequency of total CD3^+^ T cells within the CD45^+^ leukocyte population compared to the PBS control group (Figure 1). This expansion was not limited to a single lineage; both CD4^+^ helper T cells and CD8^+^ cytotoxic T cells were significantly elevated in proportion. Specifically, the 14-day group showed marked elevation in CD4^+^ percentages (p < 0.05) and CD8^+^ percentages (p < 0.05) relative to controls. The 4-day pretreatment group displayed an intermediate trend, suggesting a duration-dependent effect of EF-2001 on systemic T-cell pool expansion or recruitment potential.

3.2. Fourteen-Day EF-2001 Pretreatment Promoted a Th1-Skewed Cytokine Profile

We next investigated the functional phenotype of these expanded T cells by measuring cytokine secretion following ex vivo PMA/ionomycin stimulation. The cytokine profile of the control group was characterized by moderate levels of both Th1 and Th2 cytokines. In contrast, the 14-day EF-2001 pretreatment group demonstrated a distinct Th1 polarization. As shown in Figure 2, secretion of IFN-γ, the hallmark Th1 cytokine essential for antiviral defense, was significantly enhanced in the 14-day group compared to controls (p < 0.01). Concurrently, levels of Th2-associated cytokines, including IL-4 and IL-10, were significantly reduced (p < 0.05). IL-2 production was also unexpectedly lower in the 14-day group, which may reflect increased consumption by the expanded T-cell population or a specific regulatory feedback loop. This high IFN-γ/low IL-4 pattern indicates that prolonged intake of heat-killed EF-2001 primes the systemic immune system toward a Type 1 response, which is theoretically favorable for intracellular pathogen clearance.

3.3. EF-2001 Did Not Reduce Lung Viral Titers or Increase Lung IFN-α at Day 2 Post-Infection

Despite the robust systemic immunomodulation observed in the spleen, we assessed whether these changes translated to improved control of viral replication in the target organ, the lung. At day 2 post-infection, which corresponds to the early exponential phase of viral replication, lung viral titers in control mice averaged approximately 4.5 log_10_ TCID_50_/mL. Neither the 4-day nor the 14-day EF-2001 pretreatment resulted in a statistically significant reduction in viral load; the difference between the EF-2001 groups and the control group was less than 0.2 log_10_ TCID_50_ (Figure 3), which is below the threshold of biological significance. Furthermore, we measured levels of IFN-α, a key Type I interferon responsible for the innate antiviral state. Lung IFN-α concentrations were comparable across all three groups, with no significant elevation observed in the EF-2001-treated mice. These results suggest that while EF-2001 effectively modulates systemic adaptive immune potential, it does not accelerate the early local innate antiviral response or viral clearance at this specific early time point.

4. Discussion

In this study, we employed a murine influenza A/H3N2 infection model to evaluate the immunomodulatory and antiviral potential of the postbiotic preparation heat-killed Enterococcus faecalis EF-2001. Our principal finding is that oral pretreatment with EF-2001, particularly over a 14-day period, significantly alters the systemic immune landscape by increasing splenic T-cell proportions and driving a Th1-skewed cytokine profile (high IFN-γ, low IL-4/IL-10). However, this systemic “immune training” did not translate into a detectable reduction in lung viral titers or enhanced pulmonary IFN-α production at day 2 post-infection. These data suggest that EF-2001 functions primarily as a systemic immunomodulator rather than a potent, direct antiviral agent in the early phase of respiratory infection.

The observed Th1 skewing aligns with established mechanisms of bacterial cell wall recognition. Heat-killed preparations like EF-2001 retain stable PAMPs, particularly peptidoglycan and lipoteichoic acid (LTA). Upon oral ingestion, these components interact with TLR2 and TLR4 on intestinal epithelial cells and dendritic cells (DCs) within the Peyer’s patches [17,18]. Activation of these receptors triggers MyD88-dependent signaling cascades, leading to the activation of NF-κB and MAP kinases, which subsequently induce the production of IL-12 and other pro-Th1 cytokines [8,19]. These gut-derived signals can influence systemic T-cell differentiation, promoting a bias toward IFN-γ-producing Th1 cells while suppressing Th2 differentiation, as evidenced by the reduced IL-4 and IL-10 levels in our study. This mechanism differs partially from live probiotics, which may also secrete metabolites like SCFAs or bacteriocins that have direct distal effects.

The discrepancy between systemic Th1 activation and the lack of lung viral reduction at day 2 is a crucial observation. Influenza viral clearance is a multi-stage process: early control (days 0–3) is dominated by innate factors (Type I interferons, neutrophils, macrophages), while adaptive clearance (days 5–8) relies on CD8^+^ CTLs and antibodies [9]. Our evaluation at day 2 likely captured the innate phase, where the systemic T-cell expansion induced by EF-2001 had not yet functionally migrated to the lung or exerted cytotoxicity. Previous studies with Lactobacillus strains showing viral reduction often assessed outcomes at later time points (day 5–7) or involved strains that potently induced Type I interferons (IFN-α/β) or Type III interferons (IFN-λ) [12,20]. For instance, Clostridium butyricum was shown to suppress influenza via the metabolite 18-HEPE inducing IFN-λ, a mechanism distinct from the cell-wall driven Th1 immunity observed here [12]. The lack of elevated IFN-α in our EF-2001 groups suggests this specific postbiotic does not strongly prime the IRF7 pathway responsible for Type I interferon surges in the lung.

Despite the lack of immediate viral suppression, the “priming” effect of EF-2001 could have significant clinical utility. The promotion of a Th1 response is generally advantageous in viral infections, as it supports CTL activity and prevents the maladaptive Th2 responses sometimes associated with severe influenza immunopathology (e.g., eosinophilic infiltration or delayed clearance) [10]. Therefore, EF-2001 might be most effective as an adjunctive vaccine adjuvant. By creating a Th1-biased systemic environment, co-administration of EF-2001 with influenza vaccines could potentially enhance the immunogenicity and protective efficacy of the vaccine, particularly in populations with immunosenescence where Th1 responses are blunted [21,22].

The safety and stability of heat-killed preparations offer distinct advantages over live probiotics. In vulnerable populations such as the elderly, neonates, or immunocompromised patients, live probiotics carry small but non-negligible risks of translocation and infection [4]. Postbiotics like EF-2001 eliminate infectivity risks while maintaining immunogenicity. Our study supports the concept that viability is not a prerequisite for immune modulation, consistent with recent ISAPP consensus definitions [4].

5. Limitations and Future Directions

Several limitations of this study warrant discussion. First, the single time-point analysis (day 2) precluded observation of the adaptive immune effector phase; it is possible that the expanded Th1 pool would accelerate viral clearance at days 5–7. Future studies should include longitudinal sampling. Second, we did not measure mucosal IgA or lung-resident memory T cells, which are critical for gut-lung axis protection. Third, the use of strong polyclonal stimulation (PMA/ionomycin) reveals maximal potential rather than antigen-specific responses; influenza-specific T-cell assays would provide more granular insight. Finally, the study was not designed to evaluate clinical endpoints such as survival or weight loss.

Future research should focus on: (1) evaluating viral kinetics at later time points (days 5, 7, 10); (2) assessing the adjuvant effect of EF-2001 when combined with influenza vaccination; (3) mechanistic studies using TLR2/4 knockout mice to confirm the signaling pathway; and (4) analyzing gut microbiota composition to determine if EF-2001 indirectly modulates the host microbiome despite being non-viable.

In conclusion, heat-killed E. faecalis EF-2001 serves as a systemic immune trainer, promoting a Th1-dominant environment. While this did not confer immediate innate antiviral protection in the lung, the established Th1 bias suggests potential utility in strategies aimed at boosting adaptive immunity or vaccine responsiveness.