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.

Shigella -Specific Immune Profiles Induced after Parenteral Immunization or Oral Challenge with Either Shigella flexneri 2a or Shigella sonnei

Although immune correlates of protection have yet to be defined for shigellosis, prior studies have demonstrated that Shigella infection provides protection against reinfection in a serotype-specific manner. Therefore, it is likely that subjects with moderate to severe disease post-oral challenge would be protected from a homologous rechallenge, and investigating immune responses in these subjects may help identify immune markers associated with the development of protective immunity.

A retrospective analysis of Shigellosis in hospitalized patients

The relevance of the study of Shigellosis is due to the fact that the pathogen is capable of invasion and the production of cyto- and neurotoxins, which leads to the development of severe inflammation and toxicosis, and with massive infection — and neurotoxicosis. Purpose: to study the clinical and laboratory features of the course of Shigellosis in hospitalized patients at the present stage. Materials and Methods: a retrospective study of 17 case histories of children aged 9 month to 17 years old, hospitalized in the infectious diseases department of a Moscow hospital in 2019 with a confirmed diagnosis of Shigellosis, was carried out. Results: depending on the season, the frequency of hospitalizations of children with Shigellosis was practically the same in autumn, winter and spring, excluding summer (35%, 35%, 30% and 0%, respectively). Children with Shigellosis under the age of 1 year were 2, 1—3 years old — 4, 3—7 years old — 5, 7—14 years old — 3, over 14 years old — 3. The diagnosis of Shigellosis was confirmed in 13 patients according to the results of bacteriological research , in 4 — according to IHR (indirect hemagglutination reaction) data (without seeding). Shigella flexneri 2a was detected in 14 children, Shigella sonnei in 2, Shigella flexneri 2a together with Shigella sonnei in 1. Most of the patients 82.4% (14/17) were admitted to the hospital in the first three days of illness (by 2.8 ± 0.05 days). Intoxication symptoms were expressed in all patients, vomiting — in 76.5% of cases (13/17), fever — in 94% (16/17), abdominal pain — in 82.4% (14/17), inflammatory impurities in feces (cloudy mucus) — in all patients, hemocolitis (streaks of blood in feces) — in all but one — 94% (16/17). In one third of patients, Shigellosis was severe — in 35% (6/17). Conclusions. Shigellosis remains relevant due to the incidence of hemocolitis, severe forms, which requires hospitalization.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells

ABSTRACT Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri. LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen. IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.