Probiotic-derived amphiphilic exopolysaccharide self-assembling adjuvant delivery platform for enhancing immune responses.

Enhancing immune response activation through the synergy of effective antigen delivery and immune enhancement using natural, biodegradable materials with immune-adjuvant capabilities is challenging. Here, we present NAPSL.p that can activate the Toll-like receptor 4 (TLR4) pathway, an amphiphilic exopolysaccharide, as a potential self-assembly adjuvant delivery platform. Its molecular structure and unique properties exhibited remarkable self-assembly, forming a homogeneous nanovaccine with ovalbumin (OVA) as the model antigen. When used as an adjuvant, NAPSL.p significantly increased OVA uptake by dendritic cells. In vivo imaging revealed prolonged pharmacokinetics of NAPSL. p-delivered OVA compared to OVA alone. Notably, NAPSL.p induced elevated levels of specific serum IgG and isotype titers, enhancing rejection of B16-OVA melanoma xenografts in vaccinated mice. Additionally, NAPSL.p formulation improved therapeutic effects, inhibiting tumor growth, and increasing animal survival rates. The nanovaccine elicited CD4+ and CD8+ T cell-based immune responses, demonstrating the potential for melanoma prevention. Furthermore, NAPSL.p-based vaccination showed stronger protective effects against influenza compared to Al (OH)3 adjuvant. Our findings suggest NAPSL.p as a promising, natural self-adjuvanting delivery platform to enhance vaccine design across applications.

Mammary γδ T cells promote IL-17A-mediated immunity against Staphylococcus aureus-induced mastitis in a microbiota-dependent manner.

Mastitis, a common disease for female during lactation period that could cause a health risk for human or huge economic losses for animals, is mainly caused by S. aureus invasion. Here, we found that neutrophil recruitment via IL-17A-mediated signaling was required for host defense against S. aureus-induced mastitis in a mouse model. The rapid accumulation and activation of Vγ4+ γδ T cells in the early stage of infection triggered the IL-17A-mediated immune response. Interestingly, the accumulation and influence of γδT17 cells in host defense against S. aureus-induced mastitis in a commensal microbiota-dependent manner. Overall, this study, focusing on γδT17 cells, clarified innate immune response mechanisms against S. aureus-induced mastitis, and provided a specific response to target for future immunotherapies. Meanwhile, a link between commensal microbiota community and host defense to S. aureus mammary gland infection may unveil potential therapeutic strategies to combat these intractable infections.

Oral Vaccination with Engineered Probiotic Limosilactobacillus reuteri Has Protective Effects against Localized and Systemic Staphylococcus aureus Infection.

Staphylococcus aureus is a Gram-positive bacterium responsible for most hospital-acquired (nosocomial) and community-acquired infections worldwide. The only therapeutic strategy against S. aureus-induced infections, to date, is antibiotic treatment. A protective vaccine is urgently needed in view of the emergence of antibiotic-resistant strains associated with high-mortality cases; however, no such vaccine is currently available. In our previous work, the feasibility of implementing a Lactobacillus delivery system for development of S. aureus oral vaccine was first discussed. Here, we describe systematic screening and evaluation of protective effects of engineered Lactobacillus against S. aureus infection in terms of different delivery vehicle strains and S. aureus antigens and in localized and systemic infection models. Limosilactobacillus reuteri WXD171 was selected as the delivery vehicle strain based on its tolerance of the gastrointestinal environment, adhesion ability, and antimicrobial activities in vitro and in vivo. We designed, constructed, and evaluated engineered L. reuteri strains expressing various S. aureus antigens. Among these, engineered L. reuteri WXD171-IsdB displayed effective protection against S. aureus-induced localized infection (pneumonia and skin infection) and, furthermore, a substantial survival benefit in systemic infection (sepsis). WXD171-IsdB induced mucosal responses in gut-associated lymphoid tissues, as evidenced by increased production of secretory IgA and interleukin 17A (IL-17A) and proliferation of lymphocytes derived from Peyer's patches. The probiotic L. reuteri-based oral vaccine appears to have strong potential as a prophylactic agent against S. aureus infections. Our findings regarding utilization of Lactobacillus delivery system in S. aureus vaccine development support the usefulness of this live vaccination strategy and its potential application in next-generation vaccine development. IMPORTANCE We systematically screened and evaluated protective effects of engineered Lactobacillus against S. aureus infection in terms of differing delivery vehicle strains and S. aureus antigens and in localized and systemic infection models. Engineered L. reuteri was developed and showed strong protective effects against both types of S. aureus-induced infection. Our findings regarding the utilization of a Lactobacillus delivery system in S. aureus vaccine development support the usefulness of this live vaccination strategy and its potential application in next-generation vaccine development.

Novel exopolysaccharide derived from probiotic Lactobacillus pantheris TCP102 strain with immune-enhancing and anticancer activities.

Probiotics are gaining attention due to their functions of regulating the intestinal barrier and promoting human health. The production of exopolysaccharide (EPS) is one of the important factors for probiotics to exert beneficial properties. This study aimed to screen exopolysaccharides-producing lactic acid bacteria (LAB) and evaluate the probiotic potential. we obtained three exopolysaccharide fractions (EPS1, EPS2, and EPS3) from Lactobacillus pantheris TCP102 and purified by a combination of ion-exchange chromatography and gel permeation chromatography. The structures of the fractions were characterized by FT-IR, UV, HPLC, and scanning electron microscopy (SEM) analysis. The Mw of EPS1, EPS2, and EPS3 were approximately 20.3, 23.0, and 19.3 kDa, and were mainly composed of galactose, glucose, and mannose, with approximate molar ratios of 2.86:1:1.48, 1.26:1:1, 1.58:1.80:1, respectively. Furthermore, SEM analysis demonstrated that the three polysaccharide fractions differ in microstructure and surface morphology. Additionally, preliminary results for immune-enhancing and anticancer activities reveal that these EPSs significantly induced the production of nitric oxide (NO), TNF-α, and IL-6 in Ana-1 cells and peritoneal macrophage cells. Meanwhile, the EPSs also significantly suppressed the proliferation of HCT-116, BCG-803, and particularly A-2780 cells. The results suggest that the three novel EPSs isolated from Lactobacillus pantheris TCP102 can be regarded as potential application value in functional food and natural antitumor drugs.

Oral Administration with Recombinant Attenuated Regulated Delayed Lysis Salmonella Vaccines Protecting against Staphylococcus aureus Kidney Abscess Formation.

Abscess formation is one of the main symptoms of Staphylococcus aureus infection. It is very important to inhibit abscess formation for preventing S. aureus persistent infection. To find a feasible solution, the live oral vaccines delivering S. aureus antigens, rEsxAB and rHlam, were constructed, which were based on the attenuated regulated delayed lysis Salmonella enterica subspecies Serovar Typhimurium strain χ11802, and the inhibiting effect on abscess formation was evaluated in mice kidneys. As the results showed, after oral administration, humoral immunity was induced via the mucosal route as the antigen-specific IgG in the serum and IgA in the intestinal mucus both showed significant increases. Meanwhile, the production of IFN-γ and IL-17 in the kidney tissue suggested that Th1/Th17-biased cellular immunity played a role in varying degrees. After challenged intravenously (i.v.) with S. aureus USA300, the χ11802(pYA3681-esxAB)-vaccinated group showed obvious inhibition in kidney abscess formation among the vaccinated group, as the kidney abscess incidence rate and the staphylococcal load significantly reduced, and the kidney pathological injury was improved significantly. In conclusion, this study provided experimental data and showed great potential for live oral vaccine development with the attenuated regulated delayed lysis Salmonella Typhimurium strains against S. aureus infection.

Oral Delivery of Novel Recombinant Lactobacillus Elicit High Protection against Staphylococcus aureus Pulmonary and Skin Infections.

Staphylococcus aureus is a leading cause of nosocomial and community-associated infection worldwide; however, there is no licensed vaccine available. S. aureus initiates infection via the mucosa; therefore, a mucosal vaccine is likely to be a promising approach against S. aureus infection. Lactobacilli, a non-pathogenic bacterium, has gained increasing interest as a mucosal delivery vehicle. Hence, we attempted to develop an oral S. aureus vaccine based on lactobacilli to cushion the stress of drug resistance and vaccine needs. In this study, we designed, constructed, and evaluated recombinant Lactobacillus strains synthesizing S. aureus nontoxic mutated α-hemolysins (HlaH35L). The results from animal clinical trials showed that recombinant Lactobacillus can persist for at least 72 h and can stably express heterologous protein in vivo. Recombinant L. plantarum WXD234 (pNZ8148-Hla) could induce robust mucosal immunity in the GALT, as evidenced by a significant increase in IgA and IL-17 production and the strong proliferation of T-lymphocytes derived from Peyer's patches. WXD234 (pNZ8148-Hla) conferred up to 83% protection against S. aureus pulmonary infection and significantly reduced the abscess size in a S. aureus skin infection model. Of particular interest is the sharp reduction of the protective effect offered by WXD234 (pNZ8148-Hla) vaccination in γδ T cell-deficient or IL-17-deficient mice. In conclusion, for the first time, genetically engineered Lactobacillus WXD234 (pNZ8148-Hla) as an oral vaccine induced superior mucosal immunity, which was associated with high protection against pulmonary and skin infections caused by S. aureus. Taken together, our findings suggest the great potential for a delivery system based on lactobacilli and provide experimental data for the development of mucosal vaccines for S. aureus.

Vaccine Composition Formulated with a Novel Lactobacillus-Derived Exopolysaccharides Adjuvant Provided High Protection against Staphylococcus aureus.

A vaccine that effectively targets methicillin-resistant Staphylococcus aureus (MRSA) is urgently needed, and has been the focus of studies by numerous research groups, but with limited success to date. Recently, our team found that exopolysaccharides derived from probiotic Lactobacilluscasei strain WXD030 as an adjuvant-formulated OVA could upregulate IFN-γ and IL-17 expression in CD4+ T cells. In this study, we developed a vaccine (termed rMntC-EPS) composed of S. aureus antigen MntC and Lactobacillus casei exopolysaccharides, which conferred high levels of protection against S. aureus infection. METHODS: Six-eight-week-old female mice were vaccinated with purified rMntC-EPS30. The immune protection function of rMntC-EPS30 was assessed by the protective effect of rMntC-EPS30 to S. aureus-induced pulmonary and cutaneous infection in mice, bacterial loads and H&E in injury site, and ELISA for inflammation-related cytokines. The protective mechanism of rMntC-EPS30 was assessed by ELISA for IgG in serum, cytokines in the spleen and lungs of vaccinated mice. In addition, flow cytometry was used for analyzing cellular immune response induced by rMntC-EPS30. For confirmation of our findings, three kinds of mice were used in this study: IL-17A knockout mice, IFN-γ knockout mice and TCRγ/δ knockout mice. RESULTS: rMntC-EPS30 conferred up to 90% protection against S. aureus pulmonary infection and significantly reduced the abscess size in the S. aureus cutaneous model, with clearance of the pathogen. The rMntC-EPS vaccine could induce superior humoral immunity as well as significantly increase IL-17A and IFN-γ production. In addition, we found that rMntC-EPS vaccination induced robust Th 17/γδ T 17 primary and recall responses. Interestingly, this protective effect was distinctly reduced in the IL-17A knockout mice but not in IFN-γ knockout mice. Moreover, in TCRγ/δ knockout mice, rMntC-EPS vaccination neither increased IL-17A secretion nor provided effective protection against S. aureus infection. CONCLUSIONS: These data demonstrated that the rMntC formulated with a novel Lactobacillus-derived Exopolysaccharides adjuvant provided high protection against Staphylococcus aureus. The rMntC-EPS vaccine induced γδ T cells and IL-17A might play substantial roles in anti-S. aureus immunity. Our findings provided direct evidence that rMntC-EPS vaccine is a promising candidate for future clinical application against S. aureus-induced pulmonary and cutaneous infection.

Exopolysaccharides from Lactobacillus kiferi as adjuvant enhanced the immuno-protective against Staphylococcus aureus infection.

Exopolysaccharides from lactic acid bacteria (LAB) have gained more attention due to their health benefits. Most research on LAB EPS focuses on antitumor and antioxidant activities. To our knowledge, the immunoadjuvant activity of LAB EPS has not been thoroughly studied. In this study, the EPS produced by Lactobacillus kiferi WXD029 were purified by ethanol precipitation and column chromatography fractionation. The molecular weight of the EPS was 3.423 × 105 Da and was mainly composed of Glu, GlcN, and GalN in a molar ratio of 3.1:1:1. In vitro, EPS could significantly enhance the proliferation and phagocytic activity as well as induce the production of NO, TNF-α, IL-1ß, and IL-6 in RAW264.7 cells. In vivo, the EPS adjuvant could increase the titers of S.aureus antigen-specific antibodies and markedly enhanced T cell proliferation. Notably, EPS adjuvant also induced a strong potential Th1, Th2 and Th17-cell mixture responses. Furthermore, immunization with S.aureus antigen plus EPS adjuvant induced a protective effect when compared with S.aureus antigen alone in murine bacteremia, pneumonia and mastitis model. Collectively, these results suggest that EPS derived from probiotic Lactobacillus kiferi strain is promising as an efficient adjuvant candidate for the prevention of S. aureus infections.

Exopolysaccharides from Lactobacillus buchneri TCP016 Attenuate LPS- and d-GalN-Induced Liver Injury by Modulating the Gut Microbiota.

Liver diseases alter the gut microbiota, but several lactic acid bacteria can reduce the degree of liver damage. The present study investigated whether Lactobacillus buchneri TCP016 reduces the degree of liver damage by modifying the gut microbiota via its exopolysaccharides (EPSs). First, it was illustrated that the main EPS (EPS016; molecular weight = 8.509 × 104 Da) comprised rhamnose, xylose, glucosamine, glucuronic acid, galactose, galacturonic acid, glucose, and mannose in molar ratios of 9.2:3.9:3.8:2.8:2.1:2.0:1.6:1.0. Our data showed that EPS016 alleviated the increase in plasma and hepatic enzyme and cytokine levels, increased superoxide dismutase and glutathione activity, and alleviated bacterial translocation to the liver and mesenteric lymph nodes in vivo. Furthermore, EPS016 ameliorated intestinal mucosal injury and gut flora dysbiosis, thereby decreasing the enrichment of Helicobacteraceae, Lachnospiraceae, and Enterobacteriaceae and increasing the abundance of Lactobacillus, Rikenellaceae, Bacteroidaceae, Bacteroidales_S24-7_group, and Prevotellaceae. These findings indicated that EPS016 inhibits lipopolysaccharides/d-galactosamine-induced liver injury and improves the modification of the gut microbiota.