Mapping the functional B-cell epitopes of Shigella invasion plasmid antigen D (IpaD).

Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.

Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

Background: The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. Methods: The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Results: Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Antisuero AA479: nuevo reactivo para la serotipificación de variantes atípicas se Shigella flexneri / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological dat

Shigella flexneri se divide en al menos 13 serotipos sobre la base de la combinación de determinantes antigénicos presentes en el antígeno O. Se identificó una nueva modificación del antígeno O con fosfoetanolamina. La presencia de este determinante antigénico (denominado E1037) es reconocida por el anticuerpo monoclonal MASF IV-1. Teniendo en cuenta la incidencia creciente de estas nuevas variantes y la dificultad en la provisión del anticuerpo monoclonal para nuestro país, se elaboró un antisuero de tipo policlonal (AA479) mediante la inmunización con un cultivo de S. flexneri Xv. La especificidad del antisuero se evaluó por aglutinación en lámina con aislamientos clínicos y cultivos de colección, con lo que quedaron representados todos los serotipos de Shigella. Los resultados obtenidos demostraron una correlación del 100% entre el antisuero AA479 absorbido y el anticuerpo monoclonal MASF IV-1. La disponibilidad del antisuero AA479 en todos los hospitales públicos de Argentina permitirá identificar los aislamientos atípicos de S. flexneri; de esta forma se podrá fortalecer la vigilancia de Shigella en nuestro país y comparar con los datos epidemiológicos a nivel global

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Septicemia por Shigella flexneri / Septicaemia by Shigella flexneri

Se reporta el primer caso de aislamiento de Shigella flexneri en sangre, en Costa Rica; se hace referencia de lo poco usual de este hallazgo y énfasis en la resistencia de esta bacteria a los antibióticos orales convencionales, y la necesidad de tratar este caso con una cefalosporina de tercera generación.

IgA secretora total e específica no colostro e leite de mães da cidade do Natal-Rio Grande do Norte, Brasil / Total and specific IgA in colostrum and milk of mothers of Natal-Rio Grande do Norte, Brasil

PURPOSE: To determine the concentration of total secretory IgA and evaluate the repertoire of IgA antibodies to enteropathogenic Escherichia coli and Shigella flexneri antigens in colostrums and milk from mothers in Natal, RN. METHODS: The sample was constituted by 22 healthy clinically women whose babies were born at public hospital in Natal, RN. To determine total secretory IgA a radial immunedifusion tecnique (Mancini et al, 1965), was employed and to detect specific antibodies, immuneenzimatic assays, ELISA was used. RESULTS: The median values of total secretory IgA concentration presented individual variations with high levels in colostrums samples, decreasing during lactation, it was observed a p < 0.001 among the samples from the first day of lactation, to the thirtieth for total IgA concentration. All the donators present in colostrum and milk specific antibodies to Escherichia coli enteropathogenic (EPEC) and Shigella flexneri with titles higer in colostrum. There was parallel and directional pattern between total IgA and IgA anti-EPEC and Shegella flexneri, during period. CONCLUSION: The concentrations of total SIgA and specific antibodies to enteropathogenic Escherichia coli and Shigella flexneri in colostrums and milk in our study do not differ from others accomplished among populations with the same social and econimic features, stressing the importance of human milk as a protector agent against pathogens.