With the escalating threat of viral infections, the development of effective vaccine strategies has become critically important (Piot et al., 2019). Although a variety of vaccines are already in widespread use, their immunogenicity remains suboptimal, particularly in immunocompromised or high-risk populations (Meng et al., 2022). Therefore, improving vaccine efficacy, especially in these high-risk groups, is of paramount importance.

In recent years, research has shown that probiotics, when used as immunoadjuvants, can enhance vaccine-induced immune responses by modulating both gut and systemic immunity (Lin et al., 2021; Peroni and Morelli, 2021). Probiotics from the Lactobacillus genus, including Lactobacillus brevis, have been demonstrated to regulate immune responses through mechanisms such as cytokine production (Yin et al., 2023; Zielińska et al., 2019), T cell responses (Karimi et al., 2009; Maassen et al., 2000), and antigen presentation (Prado Acosta et al., 2021), thereby enhancing immune protection.

Lactobacillus brevis ZG2488 (hereafter referred to as L. brevis ZG2488) is a novel strain isolated by our research group from the feces of healthy humans (Cao et al., 2025). In preliminary studies, we found that ZG2488 exhibits significant probiotic characteristics, including resistance to artificial gastric juice and bile salts, high adhesion ability, and antimicrobial activity. Further research has shown that ZG2488 can enhance nitric oxide production and modulate the expression of pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in macrophages, thereby positively influencing immune responses (Cao et al., 2025). Despite its demonstrated immunomodulatory effects, the role of ZG2488 in enhancing the immunogenicity of the SARS-CoV-2 vaccine remains understudied.

This study hypothesizes that Lactobacillus brevis ZG2488, through its immunomodulatory properties, can enhance the immune response to the SARS-CoV-2 vaccine, particularly in mucosal and cellular immunity, thus improving the overall vaccine efficacy. To test this hypothesis, we assessed the effects of ZG2488 on the immune response in a mouse model, specifically evaluating its ability to enhance the immunogenicity of the SARS-CoV-2 vaccine (AdC68-Delta-S). We focused on examining the impact of ZG2488 on local mucosal immune responses, systemic cellular immunity, and immune cell infiltration patterns, and further explored its potential molecular mechanisms through transcriptomic analysis.

This study aimed to investigate the impact of L. brevis ZG2488 on the immune response to the SARS-CoV-2 vaccine in mice, focusing on various immune parameters such as SARS-CoV-2-specific IgG antibodies, IFN-γ secretion, immune cell infiltration, and transcriptomic changes. Our results indicate that L. brevis ZG2488 significantly enhanced antigen-specific antibody and T-cell responses, providing evidence for its immunomodulatory potential in improving vaccine efficacy (Redenti et al., 2024; Ryan et al., 2025; Zhu et al., 2025). Beyond mechanistic insights, our findings suggest the potential clinical relevance of ZG2488 as a candidate vaccine adjuvant. Orally administered ZG2488 was associated with enhanced mucosal humoral immunity (elevated BALF IgG) and systemic Th1-cell-mediated immunity (increased IFN-γ production). This dual activity may be advantageous against respiratory pathogens such as SARS-CoV-2, where immunity at the mucosal site of entry could contribute to overall protection. Further clinical studies are needed to evaluate the translational potential of ZG2488-adjuvanted vaccination in humans.

Lactobacillus brevis ZG2488 preferentially enhanced local mucosal immunity in the respiratory tract, as evidenced by a significant increase in SARS-CoV-2-specific IgG antibodies in bronchoalveolar lavage fluid (BALF), while serum IgG showed an upward trend but did not reach statistical significance. This result is consistent with previous studies suggesting that probiotics not only modulate systemic immunity but also enhance local immune responses, particularly in mucosal tissues (Jin et al., 2021; Ma et al., 2023; Tseng et al., 2025; Zhao et al., 2024). Lactobacilli, especially, are crucial in enhancing local immunity by promoting gut mucosal immune responses and boosting the efficacy of antigen-presenting cells, which are key for improving local vaccine immune responses (Rawling et al., 2023; van Baarlen et al., 2013; Zhang et al., 2023).

In addition to enhancing humoral immunity, L. brevis ZG2488 significantly improved the ability of splenocytes to produce IFN-γ in response to the SARS-CoV-2 vaccine, both in vivo and in vitro. IFN-γ, as a key effector cytokine in the Th1 immune response, is essential for antiviral defense and cellular immunity (Assaf et al., 2017; Lei et al., 2024; Rosloniec et al., 2002). The upregulation of IFN-γ production by L. brevis ZG2488 suggests that the probiotic enhances the ability to clear the virus, potentially improving the immune response to SARS-CoV-2. Transcriptomic analysis further revealed the molecular basis of this enhancement, highlighting the upregulation of genes involved in the JAK–STAT signaling pathway (Ifng, Stat1, Stat2, Jak2) and the antigen processing and presentation pathway (Tap1, MHC-II genes, Hsps). These findings align with other studies on Lactobacilli’s effects on immune regulation, confirming their role in enhancing immune function through activation of key immune signaling pathways (Ding et al., 2017; Ji et al., 2025). We believe that the existing data provides strong support for our mechanistic conclusions. However, due to current limitations in experimental conditions and resources, we were unable to conduct the relevant knockout validation experiments. Should the opportunity arise in the future, we would be eager to consider performing these knockout validations to further strengthen our conclusions.

Moreover, L. brevis ZG2488 displayed pleiotropic effects on innate immune signaling pathways, upregulating pro-inflammatory pathways such as IL-17, TNF, NOD-like receptor, and PI3K-Akt. Additionally, this study uncovered the pleiotropic effects of L. brevis ZG2488 on several immune signaling pathways. Interestingly, in contrast to earlier studies that found L. brevis ZG2488 downregulates TNF-α in macrophages, the current study observed an upregulation of the TNF pathway in splenocytes (Cao et al., 2025). Beyond the enrichment of the TNF signaling pathway by GSEA, we confirmed that the gene encoding the key ligand TNF-α itself was significantly upregulated in the spleens of ZG2488-treated mice compared to PBS controls (Supplementary Figure S2, p < 0.05 by t-test). This direct evidence at the gene expression level solidifies the conclusion that the TNF pathway activation is a hallmark of the immune response potentiated by L. brevis ZG2488. This apparent contradiction may be due to differences in the immune cell types involved and the specific context of the immune environment (Adami et al., 2020). While the downregulation of TNF-α in macrophages likely represents a protective effect against excessive innate inflammation, the upregulation of the TNF signaling pathway in vaccine-primed splenocytes suggests a potentiation of adaptive immunity. Many studies have demonstrated the crucial role of TNF-α in the early stages of vaccination (Maggioli et al., 2016). For example, research has shown that individuals receiving TNF inhibitors have a reduced response to the flu vaccine (Munusamy Ponnan et al., 2019). Furthermore, after COVID-19 vaccination, this pathway promotes local immune responses, helping to enhance antigen presentation, particularly in antigen processing and T cell activation (Kawakita et al., 2025). This is very important for the early immune response, aiding in the initiation of the immune response and the establishment of immune memory (Procaccini et al., 2021). Therefore, the upregulation of the TNF signaling pathway by L. brevis ZG2488 in this specific context is not necessarily contradictory but rather highlights its context-dependent immunomodulatory function. It may enhance vaccine responses under conditions that require robust T-cell activation—key processes for a strong cellular immune response (Geisen et al., 2022). This raises the possibility that L. brevis ZG2488 may act as an immunoadjuvant by upregulating TNF-α, thus stimulating a more robust inflammatory response, which in turn promotes effective antigen presentation and T-cell activation. This mechanism provides new insights into the potential of L. brevis ZG2488 as an adjuvant to enhance vaccine-induced immunity. Notably, while some probiotic strains modulate inflammation by downregulating TNF-α to prevent excessive immune activation, L. brevis ZG2488 may function differently by upregulating TNF-α to drive stronger immune responses, particularly in the early stages of immunity. These contrasting effects on TNF-α signaling reflect the strain-specific and context-dependent nature of probiotic-mediated immune modulation.

Similarly, IL-17 signaling contributes to mucosal immunity and defense against extracellular pathogens. Although dysregulated IL-17 can drive autoimmunity, its induction following vaccination supports neutrophil recruitment, barrier integrity, and antibody-mediated protection. In the context of a respiratory-targeted vaccine, this Th17-skewed response may enhance mucosal immunity in the airways (Schnoeller et al., 2014; Wu et al., 2021). The NOD-like receptor signaling pathway is involved in inflammasome activation and innate immune sensing. While aberrant NLR signaling can cause excessive inflammation, its role in vaccine adjuvancy is well-established. NLR activation promotes cytokine production and co-stimulatory molecule expression, thereby bridging innate and adaptive immunity (Higgins and Mills, 2010; Maisonneuve et al., 2014). Moreover, the PI3K-Akt signaling pathway, crucial for cell proliferation, survival, and immune responses, was also upregulated (Glaviano et al., 2023; Manning and Cantley, 2007). By activating this pathway, L. brevis ZG2488 may enhance immune cell proliferation, particularly T cells and B cells, further promoting a stronger immune defense. Importantly, the potential for immunopathology appears to be balanced by the concurrent downregulation of hyperinflammation-related pathways, such as viral protein-cytokine interactions and FcγR-mediated phagocytosis, as shown in our GSEA results (Figure 4B). This coordinated regulation—enhancing vaccine-relevant inflammation while suppressing nonspecific hyperactivation—suggests that ZG2488 promotes a focused and adaptive immune response rather than indiscriminate inflammation. Thus, the upregulation of these pathways is likely a hallmark of effective immune stimulation rather than uncontrolled inflammation. Future studies involving cytokine blockade or challenge models could further confirm the functional balance of these responses.

Conversely, L. brevis ZG2488 also selectively downregulated pathways like viral protein-cytokine receptor interaction, coronavirus disease-COVID-19, and FcγR-mediated phagocytosis. This selective downregulation could prevent excessive inflammation or resource depletion during the immune priming phase, helping to maintain a balanced immune response. These findings illustrate the complex and bidirectional nature of probiotic-mediated immune modulation, where probiotics like L. brevis ZG2488 may enhance immune responses while preventing potential harmful overactivation of certain immune pathways.

CIBERSORT analysis revealed that L. brevis ZG2488 modulates immune cell infiltration in the spleen, increasing the proportion of memory CD4⁺ T cells, Th1 cells, and DCs, while decreasing macrophages, CD4⁺ Tfh cells, monocytes, and γδ T cells. The increased DCs correlate with upregulated antigen presentation genes, which support the initiation of adaptive immune responses (Hilligan and Ronchese, 2020). The increased presence of memory CD4⁺ T and Th1 cells is indicative of a strengthened Th1 response, suggesting potential for long-term protection. The reduction in macrophages, monocytes, and Tfh cells may reflect an optimization of the immune response, suppressing overactive or potentially pro-inflammatory cell populations, thereby preventing excessive inflammation or adverse immune reactions. Lactobacilli are known to modulate immune cell infiltration in similar ways, enhancing the activity of memory T cells and Th1 cells while suppressing the overactivation of macrophages and γδ T cells to avoid excessive immune activation (Finamore et al., 2019; Liu et al., 2014; Min et al., 2023).

In summary, Lactobacillus brevis ZG2488 significantly enhanced the immune response to the SARS-CoV-2 vaccine in mice. This effect is achieved through several mechanisms: enhancing antigen presentation, modulating immune cell activity, promoting the secretion of protective cytokines (particularly IFN-γ), regulating immune signaling networks, and reshaping immune cell composition (e.g., increasing protective cells like DCs, Th1 cells, and memory CD4⁺ T cells). These findings underscore the important role of probiotics in enhancing vaccine-induced immunity, particularly through their immunomodulatory effects. We would like to emphasize that this study focused on the peak effector phase at 7 days post-boost to optimally capture the immunomodulatory effects of the probiotic, while future investigations incorporating additional timepoints will be essential to delineate the kinetics of immune response initiation, peak, and memory formation following ZG2488-adjuvanted vaccination.

We acknowledge that the sample size (n = 5 per group) is a limitation, although it meets the standard for preliminary mouse studies. However, the consistent and robust effects observed across bodily fluid, cellular, and transcriptome analyses, coupled with rigorous statistical corrections, strengthen our confidence in the conclusions. Future studies utilizing larger cohorts will be valuable for further validating these findings and exploring more subtle effects. Despite these promising results, one limitation of this study is the absence of a control group that received neither antibiotic nor probiotic treatment but was only vaccinated. Consequently, our findings specifically define the immune-enhancing effects of L. brevis ZG2488 within an antibiotic-perturbed microbiome background. It should also be noted that all mice in this study underwent antibiotic pretreatment to facilitate probiotic intervention. While this approach ensured that the observed immunomodulatory effects could be attributed to the action of L. brevis ZG2488 under controlled conditions, it prevented us from determining the independent impact of microbiota depletion itself on the vaccine response, as well as the potential influence of the probiotic mediated through gut microbiota. Future studies will include a non-antibiotic-treated control group to comprehensively dissect the respective contributions of the microbiome, antibiotic pretreatment, and probiotic intervention to vaccine-induced immune responses, and to clarify the specific roles of the microbiome and the probiotic in vaccine efficacy. Furthermore, while this study provides a foundational characterization of the novel strain L. brevis ZG2488, future research would benefit from direct comparisons with well-established probiotic adjuvants, such as LPG. Our group has previously demonstrated the efficacy of LPG in enhancing vaccine-induced immune responses in vivo (Xu et al., 2021). A dedicated comparative study, designed specifically to benchmark the efficacy of ZG2488 against LPG and other documented strains, would offer valuable insights into their relative potency and potential synergistic effects. Such research would significantly advance our understanding of the structure–activity relationships and precise mechanisms of action of different probiotic strains within the immune system. Also, while our study demonstrates enhanced immunity through antibody and IFN-γ ELISpot assays supported by transcriptomic mechanistic insights, it does not fully explore all immune functional outcomes. Future studies should include additional assays such as flow cytometry or viral neutralization to further define L. brevis ZG2488’s immunomodulatory scope. Finally, it is important to note that while our transcriptomic and immune deconvolution analyses provide a comprehensive and multi-faceted view of the immunomodulatory effects of L. brevis ZG2488, they are inherently correlative and descriptive. The causal mechanisms underlying these observations remain to be formally established. However, the strong coherence between the gene expression signatures, pathway activities, and functional immune outcomes (e.g., IFN-γ production) lends strong support to our model. Future studies employing mechanistic approaches, such as adoptive cell transfer or antibody-mediated neutralization of key cytokines like IFN-γ will be essential to definitively prove causal relationships and elucidate the precise molecular mechanisms by which L. brevis ZG2488 enhances vaccine immunity. These considerations will help optimize the therapeutic use of probiotics in vaccine adjuvant strategies.

This study demonstrates that supplementation with Lactobacillus brevis ZG2488 significantly potentiates the immunogenicity of the SARS-CoV-2 AdC68-Delta-S vaccine in mice through multifaceted immunomodulatory mechanisms. Mechanistically, ZG2488 orchestrated a dual enhancement of localized and systemic immunity by (1) augmenting mucosal humoral defenses in the respiratory tract, evidenced by elevated SARS-CoV-2-specific IgG in BALF; (2) amplifying systemic cellular immunity via robust IFN-γ production by splenocytes; (3) globally reprogramming immune signaling networks, with transcriptomic profiling revealing targeted upregulation of the IFN-γ-JAK–STAT axis and antigen presentation pathways, synergistic activation of pro-inflammatory pathways (IL-17, TNF, NLR, PI3K-Akt), and selective downregulation of antiviral/immune exhaustion pathways (viral protein–cytokine interactions, COVID-19-related genes, FcγR-mediated phagocytosis); and (4) remodeling splenic immune architecture through expansion of memory CD4⁺ T cells, Th1 subsets, and dendritic cells, coupled with contraction of macrophages, Tfh cells, monocytes, and γδ T cells. Although ZG2488 shows promise in enhancing vaccine-induced immune responses, this study did not assess its effects on clinically relevant outcomes such as protection from viral challenge, longevity of immune memory, or neutralizing antibody activity. Thus, while ZG2488 exhibits immunomodulatory properties supportive of adjuvant function, future studies are necessary to confirm its clinical utility in improving protective efficacy, sustaining immunological memory, and eliciting potent neutralizing antibody responses.