Evaluation of the prophylactic effect of egg yolk antibody (IgY) produced against the recombinant protein containing IpaD, IpaB, StxB, and VirG proteins from Shigella.

INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10 mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82 kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5 mg/ml of egg yolk and the purity of the extracted IgY was over 90 %. The results of the ELISA showed that at least 19 ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10 mg/kg IgY leads to 80 % survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.

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.

Effect of Non-Rotavirus Enteric Infections on Vaccine Efficacy in a ROTASIIL Clinical Trial.

This study examined the relative proportion of enteric pathogens associated with severe gastroenteritis (GE) among children younger than 2 years in a phase III efficacy trial of the ROTASIIL® vaccine in India, evaluated the impact of co-infections on vaccine efficacy (VE), and characterized the association between specific pathogens and the clinical profile of severe GE. Stored stool samples collected from cases of severe GE in the phase III trial were tested by quantitative polymerase chain reaction using TaqMan™ Array Cards. Etiology was attributed by calculating the adjusted attributable fraction (AF) for each pathogen. A test-negative design was used to estimate VE. The pathogens with the highest AFs for severe diarrhea were rotavirus (23.5%), adenovirus 40/41 (17.0%), Shigella spp./enteroinvasive Escherichia coli, norovirus GII, enterotoxigenic E. coli, and Cryptosporidium spp. A considerable proportion of the disease in these children could not be explained by the pathogens tested. Severe GE cases associated with rotavirus and Shigella spp. were more likely to have a longer duration of vomiting and diarrhea, respectively. Cases attributed to Cryptosporidium spp. were more severe and required hospitalization. In the intention-to-treat population, VE was estimated to be 43.9% before and 46.5% after adjustment for co-infections; in the per-protocol population, VE was 46.7% before and 49.1% after adjustments. Rotavirus continued to be the leading cause of severe GE in this age group. The adjusted VE estimates obtained did not support co-infections as a major cause of lower vaccine performance in low- and middle-income countries.

Development of a visual Adhesion/Invasion Inhibition Assay to assess the functionality of Shigella-specific antibodies.

Introduction: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella's unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level. Objectives and methods: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix. Results: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner. Discussion: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.

Shigella Vaccines: The Continuing Unmet Challenge.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

Gastroenteritis por Shigella spp. en la comunidad de General Roca, Río Negro

En la Ciudad de General Roca se ha informado un aumento en la frecuencia de diarreas agudas durante los meses cálidos, cuyas causas aún no son conocidas. En este sentido se propuso evaluar la prevalencia de bacterias enteropatógenas de individuos con síndrome diarreico agudo y georreferenciar los casos (Programa ArcMap con Datum en Campo Inchauspey WGS84). Entre agosto 2011 y junio 2012 se procesaron 616 muestras. Los casos fueron georreferenciados según los tanques de abastecimiento de agua potable en la ciudad (tanque de almacenamiento zona alta, tanque de almacenamiento zona baja y tanque de almacenamiento JJ Gómez; todos abastecidos con acueductos provenientes del Río Negro, mientras que en época estival el tanque zona alta utiliza también el canal de riego). En 172/616 muestras (28%) fueron caracterizadas las bacterias enteropatógenas que correspondieron: 29/172 (17%) a Salmonella spp., 12/172 (7%) a Campylobacter spp., 7 /172 (4%) a Escherichia coli y 124/172 (72%) a Shigella spp. (x2 67,16 p < 0,0001; Odds ratio 6,7; IC 95% =4-10,7). De los serogrupos de Shigella spp. correspondieron 122/124 (98,4%) a Shigella flexneri y 2/124 (1,6%) a Shigella sonnei. La caracterización de los serotipos de Shigella flexneri correspondió 2/122 (1,6%) a Shigella flexneri serotipo 1, 5/122 (4,1%) a Shigella flexneri serotipo 2, 14/122 (11,5%) a Shigella flexneri AA479, 9/122 (7,4%) a Shigella flexneri sin serotipificar y finalmente 92/122 (75,4%) a Shigella flexneri serotipo 3 (x2 63,02 p < 0,0001; Odds ratio 10,4; IC 95%= 5,3-17). Shigella flexneri serotipo 3 se aisló con mayor frecuencia en los menores de 5 años (p=0,0007) respecto a los otros intervalos etarios.

A growing number of acute diarrheas have been reported in the City of General Roca during the summer months, and the causes are still unknown. Therefore, the aim of this study was to analyze the prevalence of enteropathogenic bacteria from individuals with acute diarrheic syndrome and georeference the cases (Programme ArcMap with Datum in Inchauspe y WGS84 Field). From August 2011 to June 2012, 616 samples were processed. The cases were georeferenced according to the drinking water storage tanks in the city (high region storage tank, low region storage tank and JJ Gómez storage tank; all supplied by aqueducts from the Río Negro River, and the high region tank also supplied by the irrigation channels during the summer period). The enteropathogenic bacteria were found in 172/616 samples (28%) which corresponded: 29/172 (17%) to Salmonella spp., 12/172 (7%) to Campylobacter spp., 7 /172 (4%) to Escherichia coli and 124/172 (72 %) to Shigella spp. (x2 67.16 p < 0.0001; Odds ratio 6.7; IC 95% = 4-10.7). From the serogroup of Shigella spp., 122/124 (98.4%) corresponded to Shigella flexneri and 2/124 (1.6%) to Shigella sonnei. The detection of Shigella flexneri serotypes corresponded 2/122 (1.6%) to Shigella flexneri serotype 1, 5/122 (4.1%) to Shigella flexneri serotype 2, 14/122 (11.5%) to Shigella flexneri AA479, 9/122 (7.4%) to Shigella flexneri without serotyping, and finally 92/122 (75.4%) to Shigella flexneri serotype 3 (x2 63.02 p < 0.0001; Odds ratio 10.4; IC 95%= 5.3-17). Shigella flexneri serotype 3 was isolated more often in children under 5 years old (p=0.0007), in relation to the other age groups.

Diferença de patogenicidade entre Escherichia coli enteroinvasora e Shigella flexneri em modelo experimental de infecção intestinal / Diference in pathogenicity between enteroinvasive Escherichia coli and Shigella flexneri in an experimental model of intestinal infection

Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria "driblando" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em cocultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella...

Biópsia jejunal em pacientes infectados pelo virus da imunodeficiencia humana: valor diagnóstico em infecções por Salmonella sp, Shigella sp e Campylobacter sp / Jejunal biopsy in patients infected with human immunodeficiency virus: diagnostic value in infections by Salmonella, Campylobacter sp and Shigella sp

Trinta e dois pacientes infectados pelo Vírus da Imunodeficiência Humana (HIV) foram submetidos a biópsia "per os" de jejuno, com subseqüente cultivo de aspirado e fragmento de mucosa para bactérias do gênero Salmonella, Shigella e Campylobacter. Foi avaliado o rendimento destes cultivos frente a coproculturas realizadas simultaneamente. Os pacientes foraro divididos em três grupos, de acordo com o estágio da infecção pelo HIV e pela presença ou não de sindrome diarréica. Houve crescimento de Shigella sp (1 paciente) e Campylobacter sp (2 pacientes) no grupo de indivíduos com imunodeficiência e síndrome diarréica, além de Campylobacter sp em um indivíduo com imunodeficiência e sem diarréia. Estas bactérias foram identificados em fezes, com todos os materiais jejunais negativos para os microorganismos em estudo. Conclui-se que o cultivo de material jejunal nesta amostra, não aumentou a capacidade diagnóstica de infecção por estes gêneros bacterianos obtida com coproculturas.