Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

The Role of Gut Microbiota and Innate Immune Response in an Autoimmune Pancreatitis Model.

BACKGROUND: Although the involvement of intestinal microbiota in innate immunity has been reported recently, the pathogenicity of autoimmune pancreatitis (AIP) remains unclear. This study aimed to investigate whether probiotics ameliorate inflammation in AIP through interactions with innate immunity. MATERIALS AND METHODS: The AIP mouse model was generated by intraperitoneal administration of Escherichia coli to C56BL/6 female mice. Alterations in the intestinal microbiota in the AIP group were evaluated using high-throughput sequencing. Peritoneal macrophages (PMs) were collected and cocultured in vitro with Lactobacillus gasseri (LG) or ligands of Toll-like receptors (TLRs). LG was administered intraperitoneally to AIP model mice, and pancreatitis activity was evaluated to examine the ameliorative effects of LG. RESULTS: In the AIP model mice, inflammation was significantly induced in the pancreas, and the intestinal microbiota was altered with decreased LG. Antimicrobial treatment suppressed pancreatitis. In vitro, E. coli stimulation increased inflammatory cytokine expression, which was significantly decreased when the LG or TLR7 ligand was cocultured with PMs. Intraperitoneal administration of LG to AIP model mice significantly suppressed pancreatitis. CONCLUSION: The mouse model demonstrated the involvement of intestinal microbiota in pancreatitis, and LG administration suppressed pancreatitis, possibly through TLR7 signaling in PMs. LG may be a helpful probiotic for treating AIP.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice.

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model.

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Ex Vivo Immunomodulatory Effects of Lactobacillus-, Lacticaseibacillus-, and Bifidobacterium-Containing Synbiotics on Human Peripheral Blood Mononuclear Cells and Monocyte-Derived Dendritic Cells in the Context of Grass Pollen Allergy.

Sensing of the intestinal microbiota by the host immune system is important to induce protective immune responses. Hence, modification of the gut microbiota might be able to prevent or treat allergies, mediated by proinflammatory Th2 immune responses. The aim was to investigate the ex vivo immunomodulatory effects of the synbiotics Pollagen® and Kallergen®, containing the probiotic bacterial strains Lactobacillus, Lacticaseibacillus and Bifidobacterium, in the context of grass pollen allergy. Peripheral blood mononuclear cells (PBMCs) from grass pollen-allergic patients and healthy controls were stimulated with grass pollen extract (GPE) and synbiotics and Gata3 expression and cytokine secretion analyzed. Monocyte-derived dendritic cells (MoDCs) cells were matured in the presence of GPE and synbiotics, co-cultured with autologous naïve T cells and maturation markers and cytokine secretion analyzed. GPE stimulation of PBMCs from grass pollen-allergic patients resulted in a significant higher production of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 compared to healthy controls. Gata3+CD4+ T cell induction was independent of the allergic status. The synbiotics promoted IL-10 and IFN-γ secretion and downregulated the GPE-induced Th2-like phenotype. Co-culturing naïve T cells with MoDCs, matured in the presence of GPE and synbiotics, shifted the GPE-induced Th2 cytokine release towards Th1-Th17-promoting conditions in allergic subjects. The investigated synbiotics are effective in downregulating the GPE-induced Th2 immune response in PBMCs from grass pollen-allergic patients as well as in autologous MoDC-T cell stimulation assays. In addition to increased IL-10 release, the data indicates a shift from a Th2- to a more Th1- and Th17-like phenotype.

Microbial-driven preterm labour involves crosstalk between the innate and adaptive immune response.

There has been a surge in studies implicating a role of vaginal microbiota in spontaneous preterm birth (sPTB), but most are associative without mechanistic insight. Here we show a comprehensive approach to understand the causative factors of preterm birth, based on the integration of longitudinal vaginal microbiota and cervicovaginal fluid (CVF) immunophenotype data collected from 133 women at high-risk of sPTB. We show that vaginal depletion of Lactobacillus species and high bacterial diversity leads to increased mannose binding lectin (MBL), IgM, IgG, C3b, C5, IL-8, IL-6 and IL-1ß and to increased risk of sPTB. Cervical shortening, which often precedes preterm birth, is associated with Lactobacillus iners and elevated levels of IgM, C3b, C5, C5a and IL-6. These data demonstrate a role for the complement system in microbial-driven sPTB and provide a scientific rationale for the development of live biotherapeutics and complement therapeutics to prevent sPTB.

Effects of Loigolactobacillus coryniformis K8 CECT 5711 on the Immune Response of Elderly Subjects to COVID-19 Vaccination: A Randomized Controlled Trial.

Elderly people are particularly vulnerable to COVID-19, with a high risk of developing severe disease and a reduced immune response to the COVID-19 vaccine. A randomized, placebo-controlled, double-blind trial to assess the effect of the consumption of the probiotic Loigolactobacillus coryniformis K8 CECT 5711 on the immune response generated by the COVID-19 vaccine in an elderly population was performed. Two hundred nursing home residents >60 yrs that had not COVID-19 were randomized to receive L. coryniformis K8 or a placebo daily for 3 months. All volunteers received a complete vaccination schedule of a mRNA vaccine, starting the intervention ten days after the first dose. Specific IgG and IgA antibody levels were analyzed 56 days after the end of the immunization process. No differences between the groups were observed in the antibody levels. During the intervention, 19 subjects had COVID-19 (11 receiving K8 vs. 8 receiving placebo, p = 0.457). Subgroup analysis in these patients showed that levels of IgG were significantly higher in those receiving K8 compared to placebo (p = 0.038). Among subjects >85 yrs that did not get COVID-19, administration of K8 tended to increase the IgA levels (p = 0.082). The administration of K8 may enhance the specific immune response against COVID-19 and may improve the COVID-19 vaccine-specific responses in elderly populations.

Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

Probiotic Interventions Alleviate Food Allergy Symptoms Correlated With Cesarean Section: A Murine Model.

Delivery by cesarean section (CS) is linked to an increased incidence of food allergies in children and affects early gut microbiota colonization. Furthermore, emerging evidence has connected disordered intestinal microbiota to food allergies. Here, we investigated the impact of CS on a rat model for food allergy to ovalbumin (OVA). Rats delivered by CS were found to be more responsive to OVA sensitization than vaginally born ones, displaying a greater reduction in rectal temperature upon challenge, worse diarrhea, and higher levels of OVA-specific antibodies and histamine. 16S rRNA sequencing of feces revealed reduced levels of Lactobacillus and Bifidobacterium in the CS rats. Preventative supplementation with a probiotic combination containing Lactobacillus and Bifidobacterium could protect CS rats against an allergic response to OVA, indicating that the microbiota dysbiosis contributes to CS-related response. Additionally, probiotic intervention early in life might help to rebuild aberrant Th2 responses and tight junction proteins, both of which have been linked to CS-related high allergic reactions. Taken together, this study shows that disordered intestinal microbiota plays an essential role in the pathogenesis of food allergy mediated by CS. More importantly, interventions that modulate the microbiota composition in early life are therapeutically relevant for CS-related food allergies.

Abundance of Lactobacillus in porcine gut microbiota is closely related to immune response following PRRSV immunization.

Increasing evidence shows that gut microbiota plays a critical role in host immune system development and immune regulation, thus the composition of gut microbiota may affect how individuals respond to immunizations. Currently, little evidence is available on the correlation between porcine gut microbiota and vaccine immune response. Here, we investigated the influence of gut microbiota on immune response in pigs to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Based on the antibody levels for PRRSV, the immunized pigs were divided into three groups (high, low, and others), and followed by virulent PRRSV challenge. The comprehensive analysis of microbial composition revealed that gut microbiota was similar in the richness and diversity among different groups before immunization. After immunization, the richness and diversity of gut microbial community in the high group were still similar to the low group, although there was a decrease in community diversity overtime. Interestingly, the antibody titer was positively correlated with the abundance of Lactobacillus in gut microbiota in immunized pigs. Further analysis indicated that gut microbial composition might be correlated to the clinical parameters such as body weight and rectal temperature after virus challenge. Taken together, our findings suggest that certain specific members of gut microbiota, such as Lactobacillus may serve as a mechanism for regulating the immune response following immunization in pigs.

Improvement of Myelopoiesis in Cyclophosphamide-Immunosuppressed Mice by Oral Administration of Viable or Non-Viable Lactobacillus Strains.

Myelosuppression is the major dose-limiting toxicity of cancer chemotherapy. There have been many attempts to find new strategies that reduce myelosuppression. The dietary supplementation with lactic acid bacteria (LAB) improved respiratory innate immune response and the resistance against respiratory pathogens in immunosupressed hosts. Although LAB viability is an important factor in achieving optimal protective effects, non-viable LAB are capable of stimulating immunity. In this work, we studied the ability of oral preventive administration of viable and non-viable Lactobacillus rhamnosus CRL1505 or L. plantarum CRL1506 (Lr05, Lr05NV, Lp06V or Lp06NV, respectively) to minimize myelosuppressive and immunosuppressive effects derived from chemotherapy. Cyclophosphamide (Cy) impaired steady-state myelopoiesis in lactobacilli-treated and untreated control mice. Lr05V, Lr05NV and Lp06V treatments were the most effective to induce the early recovery of bone marrow (BM) tissue architecture, leukocytes, myeloid, pool mitotic and post-mitotic, peroxidase positive, and Gr-1Low/High cells in BM. We selected the CRL1505 strain for being the one capable of maintaining its myelopoiesis-enhancing properties in its non-viable form. Although the CRL1505 treatments do not modify the Cy ability to induce apoptosis, both increased the incorporation of BrdU in BM cells. Consequently, Lr05NV and Lr05V treatments were able to promote early recovery of LSK cells (Lin-Sca-1+c-Kit+ cells), multipotent progenitors (Lin-Sca-1+c-Kit+CD34+ cells), and myeloid cells (Gr-1+Ly6G+Ly6C- cells) with respect to the untreated Cy control. In addition, these treatments were able to increase the frequency of IL17A-producing innate lymphoid cells in the intestinal lamina propria (IL-17A+RORγt+CD4-NKp46+ cells) after Cy injection. These results were correlated with an increase in the IL-17A serum levels, a GM-CSF high expression and a CXCL12 lower expression in BM. Therefore, both Lr05V and Lr05NV treatments are able to activate beneficially the IL-17A/GM-CSF axis and accelerate the recovery of Cy-induced immunosuppression by increasing BM myeloid precursors. We demonstrated for the first time the beneficial effect of CRL1505 strain on myelopoiesis affected by a chemotherapeutic drug. Furthermore, Lr05NV could be a good and safe resource for reducing chemotherapy-induced leukopenia. The results are a starting point for future research and open up broad prospects for future applications of the immunobiotics.

In Ovo and Oral Administration of Probiotic Lactobacilli Modulate Cell- and Antibody-Mediated Immune Responses in Newly Hatched Chicks.

There is some evidence that lactobacilli can strengthen the immune system of chickens. This study evaluated the effects of in ovo and oral administration of a lactobacilli cocktail on cytokine gene expression, antibody-mediated immune responses, and spleen cellularity in chickens. Lactobacilli were administered either in ovo at embryonic day 18, orally at days 1, 7, 14, 21, and 28 post-hatches, or a combination of both in ovo and post-hatch inoculation. On day 5 and 10 post-hatch, spleen and bursa of Fabricius were collected for gene expression and cell composition analysis. On days 14 and 21 post-hatch, birds were immunized with sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), and sera were collected on days 7, 14, and 21 post-primary immunization. Birds that received lactobacilli (107 CFU) via in ovo followed by weekly oral administration showed a greater immune response by enhancing antibody responses, increasing the percentage of CD4+ and CD4+CD25+ T cells in the spleen and upregulating the expression of interferon (IFN)-α, IFN-ß, interleukin (IL)-8, IL-13, and IL-18 in the spleen and expression of IFN-γ, IL-2, IL-6, IL-8, IL-12, and IL-18 in the bursa. These findings suggest that pre-and post-hatch administration of lactobacilli can modulate the immune response in newly hatched chickens.

Probiotic Potential of Lactobacillus Species in Allergic Rhinitis.

Since conventional allergy medication for asthma or allergic rhinitis (AR) can cause side effects which limit the patients' quality of life, it is of interest to find other forms of therapy. In particular, probiotic bacteria, such as Lactobacillus species, have shown anti-allergic effects in various mouse and human studies. For instance, administration of some Lactobacillus species resulted in nasal and ocular symptom relief and improvement of quality of life in children and adults suffering from rhinitis. Different changes in cytokine profiles, such as elevated Th1 and decreased Th2 cytokines, reduced allergy-related immunoglobulins and cell immigration have been found in both human and murine studies. Positive effects on patients like less activity limitations or fewer rhinitis episodes and longer periods free from asthma or rhinitis were also described following oral administration of Lactobacillus bacteria. However, it is still unclear how this type of lactic acid bacteria leads to changes in the immune system and thus inhibits the development of allergies or relieves their symptoms. This review gives an overview of current studies and draws conclusions concerning the usage of probiotic Lactobacillus strains in AR.

Ovalbumin-Induced Airway Inflammation Is Ameliorated in Dectin-1-Deficient Mice, in Which Pulmonary Regulatory T Cells Are Expanded through Modification of Intestinal Commensal Bacteria.

Asthma is an allergic chronic respiratory disease that affects more than 300 million people around the world. Dysbiosis of intestinal commensal microbiota influences the development of asthma. Dectin-1 (gene symbol: Clec7a), a C-type lectin receptor, plays an important role in the intestinal immune homeostasis by controlling regulatory T (Treg) cell differentiation through regulation of intestinal microbiota. However, it is not clear whether intestinal immune conditions affect immune responses in other organs. In this study, we examined the effects of Dectin-1 deficiency on allergic airway inflammation (AAI). OVA-induced AAI was attenuated in Clec7a -/- mice. Treg cells were more abundant in colonic lamina propria, mesenteric lymph nodes, and bronchoalveolar lavage fluid of Clec7a -/- mice after AAI induction. Treatment with antibiotics, but not an antifungal agent, decreased the abundance of intestinal Treg cells and aggravated the symptoms of AAI in Clec7a -/- mice. Transplantation of gut microbiota from Clec7a -/- mice into antibiotic-treated hosts increased the abundance of intestinal Treg cells and ameliorated AAI. Overcolonization by Lactobacillus murinus, a Dectin-1 signaling-regulated commensal bacterium, also promoted expansion of Treg cells in the colon and suppressed lung inflammation. Depletion of Treg cells with anti-CD25 Ab eliminated the phenotypic differences between wild-type and Clec7a -/- mice in OVA-induced AAI. These observations suggest that inhibition of Dectin-1 signaling ameliorates AAI by increasing the abundance of Treg cells in lungs through modification of intestinal commensal bacteria, suggesting a role for commensal microbiota in regulating inflammation in organs other than the intestine.

Lactobacillus Regulates Caenorhabditis elegans Cell Signaling to Combat Salmonella Infection.

Salmonella typhimurium DT104 infection causes the death of Caenorhabditis elegans, which can be prevented by certain Lactobacillus isolates. However, the molecular mechanisms of both the host response to the infection and the protection by Lactobacillus are largely unclear. The present study has investigated the life-span and gene expression of both wild-type (WT) and mutants in some key components of cell signaling in response to S. typhimurium infection and protection from Lactobacillus zeae. The results indicated that the gene expression of daf-16 in the DAF/ insulin-like growth factor (DAF/IGF) pathway, ced-3 and ced-9 in the programmed cell death (PCD) pathway, lys-7, spp-1, and abf-3 for antimicrobial peptide production, and bar-1 involved in the production of other defense molecules was all significantly upregulated when the wild-type (WT) was subjected to DT104 infection. On the contrary, the gene expression of tir-1, sek-1, and pmk-1 in the p38 mitogen-activated protein kinase (MAPK) pathway and clec-60, sod-3, and skn-1 for the production of other defense molecules was significantly suppressed by DT104. Pretreatment of the worms with L. zeae LB1 significantly upregulated the expression of almost all the tested genes except for ced-3, ced-9, abf-2, age-1, and dbl-1 compared with the nematode infected with DT104 only. Mutants defective in the cell signaling or other defense molecules of C. elegans were either more susceptible (defective in nsy-1, sek-1, pmk-1, ced-3, ced-9, skn-1, or daf-16) or more resistant (defective in age-1 or dbl-1) to DT104 infection than the WT except for the mutant defective in sod-3. Mutants defective in antimicrobial peptides (lys-7 or abf-3) were also more susceptible than the WT. In contrast, the mutant defective in spp-1 became more resistant. When all the mutants were pretreated with L. zeae LB1, five mutants that are defective in nsy-1, sek-1, pmk-1, abf-3, or lys-7 showed no response to the protection from LB1. These results suggest that L. zeae LB1 can regulate C. elegans cell signaling including the p38 MAPK pathway and downstream production of antimicrobial peptides and defense molecules to combat Salmonella infection.

The role of lactobacilli in inhibiting skin pathogens.

The human skin microbiota forms a key barrier against skin pathogens and is important in modulating immune responses. Recent studies identify lactobacilli as endogenous inhabitants of healthy skin, while inflammatory skin conditions are often associated with a disturbed skin microbiome. Consequently, lactobacilli-based probiotics are explored as a novel treatment of inflammatory skin conditions through their topical skin application. This review focuses on the potential beneficial role of lactobacilli (family Lactobacillaceae) in the skin habitat, where they can exert multifactorial local mechanisms of action against pathogens and inflammation. On one hand, lactobacilli have been shown to directly compete with skin pathogens through adhesion inhibition, production of antimicrobial metabolites, and by influencing pathogen metabolism. The competitive anti-pathogenic action of lactobacilli has already been described mechanistically for common different skin pathogens, such as Staphylococcus aureus, Cutibacterium acnes, and Candida albicans. On the other hand, lactobacilli also have an immunomodulatory capacity associated with a reduction in excessive skin inflammation. Their influence on the immune system is mediated by bacterial metabolites and cell wall-associated or excreted microbe-associated molecular patterns (MAMPs). In addition, lactobacilli can also enhance the skin barrier function, which is often disrupted as a result of infection or in inflammatory skin diseases. Some clinical trials have already translated these mechanistic insights into beneficial clinical outcomes, showing that topically applied lactobacilli can temporarily colonize the skin and promote skin health, but more and larger clinical trials are required to generate in vivo mechanistic insights and in-depth skin microbiome analysis.

Lactobacillus acidipiscis Induced Regulatory Gamma Delta T Cells and Attenuated Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis is a chronic autoimmune disease involving the central nervous system, and shows a high disability rate. Its pathogenesis is complicated, and there is no good treatment. In recent years, with in-depth studies on the regulation of gastrointestinal flora, the relationship between the mammalian immune system and the intestinal flora has been extensively explored. Changes in the composition and structure of the gastrointestinal flora can affect the characteristics and development of the host immune system and even induce a series of central nervous system inflammation events. The occurrence and development of multiple sclerosis are closely related to the continuous destruction of the intestinal barrier caused by intestinal dysbacteriosis. In this study, we analyzed Lactobacillus acidipiscis in a mouse model of experimental autoimmune encephalomyelitis (EAE). We found that the amount of L. acidipiscis in the intestinal tract was inversely proportional to the progress of EAE development. In addition, the number of CD4+ FOXP3+ regulatory T cells in the mesenteric lymph nodes of mice increased significantly after the mice were fed with L. acidipiscis, and the differentiation of CD4+ T cells to Th1 and Th17 cells was inhibited. However, the protective effect of L. acidipiscis was lost in γδ T cell-deficient mice and hence was concluded to depend on the presence of regulatory γδ T cells in the intestinal epithelium. Moreover, including L. acidipiscis enhanced the development of Vγ1+γδ T cells but suppressed that of Vγ4+γδ T cells. In summary, our results demonstrated the ability of L. acidipiscis to induce generation of regulatory γδ T cells that suppress the development of the encephalomyelitic Th1 and Th17 cells and the progress of EAE.

Stable Recombinant-Gene Expression from a Ligilactobacillus Live Bacterial Vector via Chromosomal Integration.

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the Lagilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, Lagilis La3::P eft -rnetB and Lagilis La3::P cwah -rnetB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens.IMPORTANCE Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.

Facilitating the gut brain axis by probiotic bacteria to modulate neuroimmune response on lead exposed zebra fish models

Unravelling the efficacy of gut biome has a major impact on health. An unbalanced microbiome composition is linked to many common illnesses such as gut dysbiosis, mental deformities and immunological imbalance. An optimistic influence on the gut biome can be made by consumingprobiotics. This would stimulate neuroprotection and immunomodulation intended by heavy metals pollution. Lead is a major source of neurotoxin that can induce neural deformities. Lactobacillusspecies isolated from curd were characterized to confirm its specificity. Zebra fish was reared at standard conditions and preclinical assessment on the intensity of induced neurotoxin lead was performed. The embryo toxic assay, immunomodulation effects and animal behavioural models endorsed the consequence of neurotoxicity. Different concentrations of bacterial isolate with standard antidepressant was considered for analysing the vigour of toxicity and its influence on cognitive behaviour by novel tank diving method. The restrain in the animal behaviour was also conferred by all the test samples with a decreased bottom dwelling time which was authenticated with haematology and histopathological studies. The alterations in morphology of the lymphocytes were balanced by the treated test samples. This study paves a twofold potential of probiotic as neuroprotectant and immune modulator against heavy metal toxicity.

Recent Insights on the Maternal Microbiota: Impact on Pregnancy Outcomes.

Hormonal changes during and after pregnancy are linked with modifications in the maternal microbiota. We describe the importance of the maternal microbiota in pregnancy and examine whether changes in maternal microbiotic composition at different body sites (gut, vagina, endometrium) are associated with pregnancy complications. We analyze the likely interactions between microbiota and the immune system. During pregnancy, the gastrointestinal (gut) microbiota undergoes profound changes that lead to an increase in lactic acid-producing bacteria and a reduction in butyrate-producing bacteria. The meaning of such changes needs clarification. Additionally, several studies have indicated a possible involvement of the maternal gut microbiota in autoimmune and lifelong diseases. The human vagina has its own microbiota, and changes in vaginal microbiota are related to several pregnancy-related complications. Recent studies show reduced lactobacilli, increased bacterial diversity, and low vaginal levels of beta-defensin 2 in women with preterm births. In contrast, early and healthy pregnancies are characterized by low diversity and low numbers of bacterial communities dominated by Lactobacillus. These observations suggest that early vaginal cultures that show an absence of Lactobacillus and polymicrobial vaginal colonization are risk factors for preterm birth. The endometrium is not a sterile site. Resident endometrial microbiota has only been defined recently. However, questions remain regarding the main components of the endometrial microbiota and their impact on the reproductive tract concerning both fertility and pregnancy outcomes. A classification based on endometrial bacterial patterns could help develop a microbiota-based diagnosis as well as personalized therapies for the prevention of obstetric complications and personalized treatments through nutritional, microbiotic, or pharmaceutical interventions.