Characterization of killed but metabolically active uropathogenic Escherichia coli strain as possible vaccine candidate for urinary tract infection.

BACKGROUND AND AIM: Urinary tract infection (UTI) is the second most frequent infection in human, and uropathogenic Escherichia coli is its most common cause. Although antibiotics are the standard treatment for UTI, they can cause harmful effects on gut microbiome and increase the rate of existing drug-resistant bacteria, which make the vaccine research reasonable. This study was conducted to construct a Killed but Metabolically Active (KBMA) E. coli strain, and to determine its characteristics as a possible vaccine candidate for UTI, which will be evaluated in further investigations. METHODS: The uvrB gene of uvrABC excision repair system of E.coli was deleted to construct a ΔuvrB mutant, lacking repairing system of intercross linkages between DNA strands. To construct KBMA strain, the ΔuvrB mutant was PUVA-treated, using different doses of 8-methoxypsoralen (8-MOP) followed by different doses of ultraviolet A (UVA) irradiation (365 nm), until the optimal doses of each were achieved. Then, different characteristics of the PUVA-treated E. coli (with the optimal doses) were assessed, using cell counting, colony formation assay, MTT and XTT assays, fluorescent staining, and flow cytometry. RESULTS: PUVA treatment's optimal dose for E. coli isolates was 150 ng/ml 8-MOP plus 1000 mj/cm2 UVA. While the PUVA-treated isolates had a significant decrease in cell counting, the fluorescent dying of the un-grown parts of the culture plates revealed living bacteria with bizarre shapes. Meanwhile, MTT and XTT assays demonstrated the metabolic activity of these bacteria and flow cytometry confirmed their aliveness. CONCLUSION: These PUVA-treated bacteria, with metabolic activity and proliferation inability, seem to be good enough to be tested in vitro and in vivo as a candidate for vaccine against UTI. Therefore it seems the first step toward development of a vaccine candidate is successfully done. The immunogenicity and protectivity of these treated bacteria is under evaluation.

Implications of enterotoxigenic Escherichia coli genomics for vaccine development.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality caused by diarrhea in children less than 5 years of age in low- and middle-income countries. Despite a wealth of research elucidating the mechanisms of disease, the immunological responses and vaccine development, ETEC is still relatively uncharacterized when it comes to regulation of virulence and detailed immune mechanisms. The recent emergence of next-generation sequencing now offers the possibility to screen genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. In this review, we discuss how recent findings on ETEC genomics using novel sequencing techniques will aid in finding novel protective antigens that can be used in vaccine approaches.

Characterization of enterotoxigenic Escherichia coli strains isolated from Nicaraguan children in hospital, primary care and community settings.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhoea among young children in developing countries. ETEC vaccines offer promise in reducing the burden of ETEC disease, but the development of these vaccines relies on the characterization of ETEC isolates from a variety of settings. To best reflect the full spectrum of ETEC disease in León, Nicaragua, the aim of this study was to characterize ETEC strains isolated from children with diarrhoea attending different settings (hospital, primary care clinics and in the community) and children from different age groups. We characterized ETEC isolates in terms of their colonization factors (CFs) and enterotoxins, and determined whether these factors varied with setting and age group. Diarrhoeal stool samples were obtained from children under the age of 60 months from: (1) the regional public hospital, (2) four public primary care clinics, and (3) a population-based cohort. In total, 58 ETEC-positive isolates were analysed by multiplex-PCR assays for the identification of CFs (CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS13, CS14, CS15, CS17, CS18, CS19, CS20, CS21, CS22 and CFA/I), and enterotoxins [heat-labile toxin (LT) and heat-stable variants STh and STp]. The frequency of CFs and enterotoxins was compared among the three settings and for different age groups, using Fisher's exact test or a χ(2) test. At least one CF was detected among one-half of samples; CS19 was detected among all strains in which a CF was identified, either alone or in combination with another CF. Among all CFs detected, 91.7 % were identified as members of the class 5 fimbrial family. CFs were detected more commonly among samples from infants captured in the health facility setting compared with the community setting. Overall, LT was detected among 67.2 % of samples, STh was detected among 20.7 % and both enterotoxins were detected among 12.1 %. The enterotoxin STh was detected more commonly among cases in the community, whilst a combination of STh and LT was detected more commonly among cases treated in health facilities. Our results suggest that, to protect against diarrhoeal cases associated with this E. coli pathotype in León, Nicaragua, an ETEC vaccine that effectively targets the archeotype CFA/I of the class 5 fimbrial family would be the most effective in this setting.

Design and characterization of highly immunogenic heat-stable enterotoxin of enterotoxigenic Escherichia coli K99(+).

In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99(+).

Protective immune response of bacterially-derived recombinant FaeG in piglets.

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli(ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, C83907 (K88ad, CT+, ST+). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2.

The hemolytic uremic syndrome is a life-threatening sequela that occurs after infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) or Shigella dysenteriae type 1, and Stx is responsible for initiating this syndrome. An STEC isolate can express Stx1, Stx2, or both, but antisera to Stx1 and Stx2 are not cross-neutralizing. To produce a single vaccine candidate against both toxins, we created a genetic toxoid that contained the enzymatically-inactivated StxA2 subunit and the native StxB1 subunit. We found that mice immunized with this hybrid holotoxoid, developed neutralizing anti-Stx1 and anti-Stx2 antibodies and survived challenge with 10 lethal doses of either or both toxins.