α-galactosylceramide-reactive NKT cells increase IgG1 class switch against a C. difficile polysaccharide antigen and enhance immunity against a live pathogen challenge

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increasing the ratio of IgG1/IgG2c. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18 -/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen towards Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Outer membrane vesicles derived from Salmonella Typhimurium can deliver Shigella flexneri 2a O-polysaccharide antigen to prevent Shigella flexneri 2a infection in mice

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a ( S. flexneri 2a) is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has not yet been developed. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella , due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively bio-synthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine both intranasally and intraperitoneally induced significant specific anti-Shigella LPS antibodies in the serum, with a similar trend IgA levels from vaginal secretions and fluid from bronchopulmonary lavage. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay (SBA), opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. Importance: Shigella , the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. Additionally, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella , from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

Containment of Cattle Brucellosis Outbreaks With a Small Dose of Vaccine From Strain 82

Improving the system of anti-brucellosis measures is the ultimate goal of all studies devoted to the problem of brucellosis. Cattle brucellosis outbreaks in the brucellosis-free regions in the Russian Federation indicate a premature cessation of immunization procedures and the need for their continuation. Meanwhile, there is an urgent problem of developing anti-brucellosis immunity in the adult cattle: the initial vaccination of cattle with a full dose of the vaccine from strain B. abortus 82 is fraught with post-vaccination abortions. In order to solve this problem, in the production conditions of one of the farms where the current epizootic situation for brucellosis required alternative approaches for containment of a brucellosis outbreak, a small dose of the vaccine was tested on the cattle without specific immunity. This resulted in immunogenicity, loss of abortogenicity and the presence of provocative properties of the vaccine in small doses. This allowed the detection of antibodies in animals with a hidden infection in the early stages after vaccination. Serological methods (AT, RBT and IDR with O-polysaccharide antigen), which are generally accepted in veterinary practice, were used as diagnostic tests. The reimmunization of the cows with a small dose of the vaccine from strain 82 allowed diagnostic tests to be carried out earlier after vaccination than after the use of a full dose of the vaccine, which made it possible to diagnose brucellosis in a short period of time after vaccination. The provocation of immunological reactions in latently sick animals after the vaccination with small doses, and the isolation of those who responded under the condition of supported immunity made it possible to achieve the recovery of farm animals in a short period of time, which was proved by negative indicators of the immunodiffusion reaction with O-polysaccharide antigen. Keywords: brucellosis, small doses of the vaccine, abortogenicity, immunity

Role of O-polysaccharide antigen made from B. melitensis in the differential diagnosis of brucellosis in small ruminants

Out of 2942 blood serum samples from small ruminants of 10 flocks with a natural course of brucellosis caused by B. melitensis, 322 samples reacted with both antigens in the RID, of which 90 samples only with the O-PS M antigen (from B. melitensis), only with O-PS A-antigen (from B. abortus) reactive was not revealed. In healthy sheep immunized against brucellosis with the vaccine from strain 19 according to different schemes, only the O-PS M antigen was not found to react. Reaction with O-PS A- and M-antigens was observed in animals that were immunized twice subcutaneously at a dose of 40 billion mc. - after 2 months. after revaccination (60%), as well as in those reimmunized conjunctivally at a dose of 4 billion mc. according to the background of primary immunization subcutaneously at a dose of 40 billion mc. (10%) In animals immunized once or twice conjunctivally, reacting in RID with both antigens was not detected. Out of 2432 blood serum samples of small ruminants, 10 flocks with a brucellosis problem immunized against brucellosis with a vaccine from B.abortus strain 19 according to different schemes, 151 samples (6.2%) reacted positively with both O-PS antigens in RID with both O-PS antigens, of which only 86 samples (56.9%) reacted with O-PS M-antigen. The prevalence of indications of RID with O-PS M-antigen over RID with O-PS A-antigen (O-PS antigen made from Brucellae abortus) in small ruminants in one or another flock is characteristic of infection caused by brucellae melitensis at least in the absence, at least in the presence of the fact of immunization with a vaccine from the B. abortus 19 strain. RID with O-PS M-antigen is an objective indicator of epizootic danger and is able to differentiate brucellosis (B. melitensis) in small ruminants from vaccination-induced reactions (B. abortus 19).

Outer membrane vesicles derived from Salmonella Typhimurium can deliver Shigella flexneri 2a O-polysaccharide antigen to prevent lethal pulmonary infection in mice

The threat to health from shigellosis has become quite serious in many developing countries, causing severe diarrhea. Shigella flexneri 2a (S. flexneri 2a) is the principal species responsible for this endemic disease. Although there have been multiple attempts to design a vaccine against Shigellosis, one that is effective has not yet been developed. Lipopolysaccharide (LPS) is both an essential virulence factor and a protective antigen against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively bio-synthesized in Salmonella and attached to core-lipid A by the ligase WaaL, and thus purified outer membrane vesicles (OMVs) were used as a vaccine for subsequent research. Here, we identified the expression of the heterologous polysaccharide antigen and described the isolation, characterization, and immune protection efficiency of the OMV vaccine. The expression of S. flexneri 2a did not affect the formation of Salmonella OMVs or their cytotoxicity. Furthermore, the results of the animal experiments indicated that immunization of the mice both intranasally and intraperitoneally with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in both vaginal secretions and fluid from bronchopulmonary lavage, in addition to within sera. The OMV vaccine immunized by both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay (SBA), opsonization assay, challenge experiment, and pathological analysis. The present study firstly indicates that the proposed vaccination strategy represents a novel and improved approach to control Shigellosis by the combination of bioconjugation of Salmonella glycosyl carrier lipid and OMV. In addition, the strategy of genetic manipulation described here is ideally suited for designs based on other Shigella serotypes, allowing the development of a Shigella vaccine with broad-protection.

Inactivation of GalU leads to a cell wall-associated polysaccharide defect that reduces the susceptibility of Enterococcus faecalis to bacteriolytic agents.

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose-1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis. Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal 288–298 a.a. region of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis. This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1. Interestingly, these mutants conferred reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalis. IMPORTANCE Cell wall-associated polysaccharides of bacteria are involved in various physiological characteristics. Recent studies demonstrated that the cell wall-associated polysaccharide of Enterococcus faecalis is required for susceptibility to bactericidal antibiotic agents. Here, we demonstrated that a galU mutation resulted in resistance to the enterococcal lytic bacteriocin Bac41. The galU homologue is reported to be essential for biosynthesis of species-specific cell wall-associated polysaccharides in other Firmicutes. In E. faecalis, the galU mutant lost the E. faecalis-specific cell wall-associated polysaccharide EPA (enterococcal polysaccharide antigen). The mutant also displayed reduced susceptibility to antibacterial agents and an abnormal cell morphology. We firstly demonstrated that galU was essential for EPA biosynthesis in E. faecalis, and EPA production might underlie susceptibility to lytic bacteriocin and antibiotic agents by undefined mechanism.

Comparison of management options for specific antibody deficiency

Background: Specific antibody deficiency is a primary immunodeficiency characterized by normal immunoglobulins with an inadequate response to polysaccharide antigen vaccination. This disease can result in recurrent infections, the most common being sinopulmonary infections. Treatment options include clinical observation, prophylactic antibiotic therapy, and immunoglobulin supplementation therapy, each with limited clinical data about their efficacy. Objective: This study aimed to identify whether there was a statistically significant difference in the rate of infections for patients who were managed with clinical observation, prophylactic antibiotics, or immunoglobulin supplementation therapy. Methods: A retrospective chart review was conducted. Patients were eligible for the study if they had normal immunoglobulin levels, an inadequate antibody response to polysaccharide antigen‐based vaccination, and no other known causes of immunodeficiency. Results: A total of 26 patients with specific antibody deficiency were identified. Eleven patients were managed with immunoglobulin supplementation, ten with clinical observation, and five with prophylactic antibiotic therapy. The frequency of antibiotic prescriptions was assessed for the first year after intervention. A statistically significant rate of decreased antibiotic prescriptions after intervention was found for patients treated with immunoglobulin supplementation (n = 11; p = 0.0004) and for patients on prophylactic antibiotics (n = 5; p = 0.01). There was no statistical difference in antibiotic prescriptions for those patients treated with immunoglobulin supplementation versus prophylactic antibiotics (p = 0.21). Conclusion: Prophylactic antibiotics seemed to be equally effective as immunoglobin supplementation therapy for the treatment of specific antibody deficiency. Further studies are needed in this area.

Inactivation of GalU leads the cell wall-associated polysaccharide defect to reduce the susceptibility to bacteriolytic agents in Enterococcus faecalis

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose-1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis. Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal 288–298 a.a. region of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis. This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1. Interestingly, these mutants conferred reduced susceptibility to beta-lactams, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalis.ImportanceCell wall-associated polysaccharides of bacteria are involved in various physiological characteristics. Recent studies demonstrated that the cell wall-associated polysaccharide of Enterococcus faecalis is required for susceptibility to bactericidal antibiotic agents. Here, we demonstrated that a galU mutation resulted in resistance to the enterococcal lytic bacteriocin Bac41. The galU homologue is reported to be essential for biosynthesis of species-specific cell wall-associated polysaccharides in other Firmicutes. In E. faecalis, the galU mutant lost the E. faecalis-specific cell wall-associated polysaccharide EPA (enterococcal polysaccharide antigen). The mutant also displayed reduced susceptibility to antibacterial agents and an abnormal cell morphology. We firstly demonstrated that galU was essential for EPA biosynthesis in E. faecalis, and EPA production might underlie susceptibility to lytic bacteriocin and antibiotic agents by undefined mechanism.