Fang Wang

Staphylococcus aureus is a major global health threat due to its increasing antibiotic resistance and high virulence, presenting serious challenges in healthcare. Currently, no effective vaccine exists to prevent its infections. In our study, we introduced a novel multi-epitope vaccine design method using immunoinformatics, named the “Just the Best Ones” strategy. This approach involved selecting the most promising T-cell and B-cell epitopes from 10 key proteins of S. aureus based on the pathogenic mechanism of the bacterium in the host, to create the Staphylococcus aureus Multi-Epitope Antigen (SAMEA) with optimal immunogenicity. Physicochemical and structural analyses confirmed SAMEA’s stability and suitable profiles for antigenicity, toxicity, and allergenicity. Molecular docking showed strong interactions between SAMEA and human TLR2, crucial for eliciting effective immune responses. Additionally, SAMEA was highly immunogenic and safe, generating a strong antibody response and excellent protection in mice. These results suggest that SAMEA is a promising multi-epitope vaccine candidate, highlighting the “Just the Best Ones” strategy’s effectiveness, and introducing a new direction in vaccine development against S. aureus infections. IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) is a major health threat due to its antibiotic resistance and ability to cause severe infections in humans and animals. Yet, no licensed vaccine is available. Here, we present a “Just the Best Ones” strategy that applies immunoinformatics to select the most protective epitopes from key S. aureus antigens. Using this approach, we developed a multi-epitope antigen (SAMEA) that proved safe, highly immunogenic, and protective in mice, particularly when combined with a bacterium-like particle adjuvant. These results highlight SAMEA as a promising MRSA vaccine candidate and indicate that this strategy may help guide vaccine development for other difficult pathogens. As it continues to be refined, this approach could facilitate more effective vaccines and improve the prevention of infectious diseases.

Ochratoxin A (OTA), a nephrotoxic, immunosuppressive, and potentially carcinogenic mycotoxin produced by Aspergillus and Penicillium species, poses a persistent threat to global food safety and livestock health. Although current biological detoxification strategies can degrade OTA, they often overlook its detrimental impact on vaccine efficacy, and safe probiotic candidates with dual functions of detoxification and immune restoration remain scarce. In this study, we isolated 3 OTA-degrading bacterial strains from 28 types of traditional Chinese fermented foods. Among them, Lysinibacillus macroides 38352 exhibited the highest degradation efficiency, removing 51.4% of 2.5 µg/mL OTA within 72 h at 37°C, with further increases observed over time. Mechanistic analysis revealed that the degradation was mediated not by cell surface adsorption but by secreted metabolites generated through active cellular metabolism. The strain also demonstrated excellent probiotic properties, including tolerance to pH 2–12 and 0.2% bile salts, sensitivity to 15 antibiotics (indicating no resistance), and no pathogenicity in mice. Notably, oral administration of L. macroides 38352 significantly reversed OTA-induced immunosuppression, enhancing vaccine-induced specific IgG, neutralizing antibody titers, and cytokine levels (interleukin [IL]-1β, IL-4, IL-12, IL-17, and interferon gamma), while also reducing inflammatory damage and improving growth performance in OTA-exposed broilers. Our study demonstrates that L. macroides 38352 is a dual-function probiotic capable of both OTA biodegradation and immune restoration. These findings provide a mechanistically distinct microbe-based strategy that couples mycotoxin detoxification with host immune restoration, advancing microbiome-informed interventions for sustainable livestock production and food safety. IMPORTANCE: Ochratoxin A (OTA) is a widespread food and feed contaminant known for its toxic, carcinogenic, and immune-disrupting effects, which pose serious challenges to animal health and food security. However, existing detoxification approaches rarely address the immune damage caused by OTA, and safe microbial solutions with both detoxifying and immune-supporting capabilities remain limited. This study introduces Lysinibacillus macroides 38352, a newly identified probiotic from traditional Chinese fermented food, as a dual-function candidate that not only degrades OTA efficiently through secreted metabolites but also helps repair immune function suppressed by OTA exposure. The strain shows excellent safety features and effectively improves vaccine responses and overall health in poultry. These findings highlight a novel microbe-based strategy that integrates mycotoxin detoxification with immune restoration, advancing the development of functional probiotics for microbiome-driven interventions in sustainable animal production and food safety.

Clostridium perfringens is one of the main causes of death in poultry with no vaccines approved for poultry at present. The appropriate adjuvant is critical for the development of vaccines in C. perfringens in poultry. Here, we utilized Levilactobacillus brevis for high-yielding selenium biotransformation and demonstrated that heat-inactivated nano-selenium Lactobacillus (HiSeL) is a safe, efficient, and chemically stable selenium immunopotentiator for C. perfringens vaccines. We evaluated the effectiveness of HiSeL as an immune adjuvant to modulate the efficacy of multi-epitope vaccine in mice. Subcutaneous immunization mice with HiSeL promoted high levels of specific IgG, modulated cytokine secretion, downregulated stress-related gene expression, and provided 100% protection against lethal challenge with C. perfringens. Surprisingly, we found that HiSeL can quickly and effectively induce SIgA production even by subcutaneous immunization. Transcriptome sequencing revealed the pivotal role of TGF-β and NF-κB signaling pathways in IgA immune responses in mice immunized with the HiSeL-adjuvanted multi-epitope vaccine. Collectively, our study provides proof-of-concept evidence that HiSeL functions as a potent adjuvant candidate for the multi-epitope vaccine in a murine model, offering new insights into the development of engineered postbiotic-based adjuvants.

Schmallenberg virus (SBV) is a negative-sense RNA virus transmitted by insect vectors, causing arthrogryposis-hydranencephaly syndrome in newborn ruminants. Since its discovery in Germany and the Netherlands in 2011, SBV has rapidly spread across multiple European countries, resulting in significant economic losses in the livestock industry. With the increasing global animal trade and the expanded range of insect transmission, the risk of SBV introduction into non-endemic regions is also rising. As the gold standard for serological testing, the virus neutralization test (VNT) is crucial for tracking the spread of SBV and evaluating the efficacy of vaccines. However, in non-endemic regions, the lack of local viral strains and the biosafety risks associated with introducing foreign strains pose challenges to the implementation of VNT. In this study, we employed reverse genetics techniques using vesicular stomatitis virus (VSV) to substitute the VSV G protein with the envelope glycoproteins of SBV, thereby successfully generating and rescuing the recombinant virus rVSVΔG-eGFP-SBVGPC. The recombinant virus was then thoroughly characterized in terms of SBV Gc protein expression, viral morphology, and growth kinetics. Importantly, rVSVΔG-eGFP-SBVGPC exhibited SBV-specific cell tropism and was capable of reacting with SBV-positive serum, enabling the measurement of neutralizing antibody titers. The results suggest that this recombinant virus can serve as a feasible alternative for SBV neutralization tests, with promising potential for application in serological screening and vaccine evaluation.

Trained immunity refers to the ability of trained innate immune cells to generate an immune memory that produces rapid, broad-spectrum, and long-lasting protection against heterologous stimuli. Based on the rapid and broad-spectrum protection that the peptidoglycan backbone from lactic acid bacteria, bacterium-like particles (BLPs), offers, we hypothesized that BLPs enhance protection through trained immunity. Here, we found that combining BLP with a vaccine significantly improves protective efficacy against methicillin-resistant Staphylococcus aureus (MRSA) infection, accompanied by changes in trained immunity markers. We demonstrate that BLP-induced trained immunity macrophages exhibit increased cytokine secretion and phagocytic activity in vitro. In an in vivo model, BLP confers protection against S. aureus 26003 even without specific antigens. In an ex vivo model, BLP induces increased markers of trained immunity. Transcriptome analysis suggests that BLP may induce trained immunity by activating the IL-6-JAK-STAT3 pathway through TLR2 receptor activation, thereby modulating macrophage metabolic reprogramming and function. In summary, our study establishes that BLP induction of trained immunity, along with regulated metabolic reprogramming and macrophage function, may contribute to enhancing vaccine efficacy. Our findings elucidate a novel mechanism for BLP-mediated immune enhancement, critical for the application of BLP as a vaccine vector to construct a vaccine that combines specific immune response with innate immune response.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system’s effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.