MecVax supplemented with CFA MEFA-II induces functional antibodies against 12 adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21) and 2 toxins (STa, LT) of enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) strains that produce various adhesins and one or two enterotoxins are the leading causes of children's diarrhea and travelers' diarrhea. MecVax, a multivalent ETEC vaccine candidate, consists of two proteins, an adhesin multiepitope fusion antigen (MEFA) that stimulates antibodies to the seven most important ETEC adhesins (CFA/I and CS1-CS6) and a toxoid fusion antigen which stimulates antibodies against ETEC enterotoxins (heat-labile toxin and heat-stable toxin). CFA MEFA-II, another polyvalent MEFA protein, has been demonstrated to stimulate antibodies to another five important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21). We hypothesize that MecVax coverage and efficacy can be expanded if MecVax could stimulate antibodies to all 12 adhesins. In this study, we supplemented MecVax with CFA MEFA-II, examined broad immunity to the 12 targeted ETEC adhesins and 2 ETEC toxins (STa, LT) in mice, and assessed mouse antibody functions for inhibiting the adherence of the 12 adhesins and neutralizing the enterotoxicity of 2 toxins, thus assessing the potential application of a broadly protective pan-ETEC vaccine. Mice intramuscularly immunized with MecVax and CFA MEFA-II developed robust antibody responses to the 12 ETEC adhesins and 2 toxins; furthermore, mouse serum antibodies showed functional activities against the adherence from each of the targeted adhesins and the enterotoxicity of either toxin. Data also indicated that CFA MEFA-II was antigenically compatible with MecVax. These results demonstrated that the inclusion of CFA MEFA-II further expands MecVax broad immunogenicity and protection coverage, suggesting the feasibility of developing a vaccine against all important diarrheal ETEC strains.IMPORTANCEThere are no vaccines licensed for Enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Since ETEC strains produce over 25 adhesins and 2 distinctive enterotoxins, heterogeneity is a key obstacle to vaccine development. MecVax, a multivalent ETEC vaccine candidate, induces protective antibodies against the seven most important adhesins (CFA/I and CS1-CS6) associated with two-thirds of ETEC clinical cases. However, ETEC prevalence shifts chronically and geographically, and other adhesins are also associated with clinical cases. MecVax would become a pan-ETEC vaccine if it also protects against the remaining important adhesins. This study demonstrated that MecVax supplemented with adhesin protein CFA MEFA-II induces functional antibodies against 12 important ETEC adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21), enabling the development of a more broadly protective ETEC vaccine and further validating the application of the MEFA vaccinology platform for multivalent vaccine development.

Glycan and Protein Analysis of Glycoengineered Bacterial E. coli Vaccines by MALDI-in-Source Decay FT-ICR Mass Spectrometry.

Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, such as O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Since development and quality control processes rely on such characterization, novel strategies are needed for faster and informative analysis. Here, we present a novel approach employing minimal sample preparation and ultrahigh-resolution mass spectrometry analysis for protein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Three glycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides from E. coli serotypes O2, O6A, and O25B with the genetically detoxified exotoxin A from Pseudomonas aeruginosa, were analyzed by MALDI-in-source decay (ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycan fragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage development.

Exploiting the power of OMICS approaches to produce E. coli O157 vaccines.

Enterohemorrhagic Escherichia coli (EHEC) strains are well-documented human pathogens and causative agents of diarrheal episodes and hemorrhagic colitis. The serotype O157:H7 is highly virulent and responsible for both outbreaks and sporadic cases of diarrhea. Because antibiotic treatment is contraindicated against this pathogen, development of a human vaccine could be an effective intervention in public health. In our recent Infection and Immunity paper, we applied integrated approaches of in silico genome wide search combined with bioinformatics tools to identify and test O157 vaccine candidates for their protective effect on a murine model of gastrointestinal infection. Using genomic/immunoinformatic approaches that are further described here, we categorized vaccine candidates as high, medium, and low priorities, and demonstrate that some high priority candidates were able to significantly induce Th2 cytokines and reduce EHEC colonization. Using the STRING database, we have recently evaluated the vaccine candidates and predict functional protein interactions, determining whether correlations exist for the development of a multi-subunit vaccine, targeting different pathways against EHEC O157:H7. The overall approach is designed to screen potential vaccine candidates against EHEC; however, the methodology can be quickly applied to many other intestinal pathogens.

Crystal structure of c5321: a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold.

BACKGROUND: Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. RESULTS: We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. CONCLUSIONS: The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Evaluation of the adjuvant effect of Salmonella-based Escherichia coli heat-labile toxin B subunits on the efficacy of a live Salmonella-delivered avian pathogenic Escherichia coli vaccine.

The present study evaluated the adjuvant effect of live attenuated salmonella organisms expressing the heat-labile toxin of Escherichia coli B subunit (LTB) on the efficacy of an avian pathogenic Escherichia coli (APEC) vaccine. The Asd(+) (aspartate semialdehyde dehydrogenase) plasmid pMMP906 containing the LTB gene was introduced into a Salmonella enterica Typhimurium strain lacking the lon, cpxR and asd genes to generate the adjuvant strain. Live recombinant Salmonella-delivered APEC vaccine candidates were used for this study. The birds were divided into three groups: group A, non-vaccinated controls; group B, immunized with vaccine candidates only; and group C, immunized with vaccine candidates and the LTB strain. The immune responses were measured and the birds were challenged at 21 days of age with a virulent APEC strain. Group C showed a significant increase in plasma IgG and intestinal IgA levels and a significantly higher lymphocyte proliferation response compared with the other groups. Upon challenge with the virulent APEC strain, group C showed effective protection whereas group B did not. We also attempted to optimize the effective dose of the adjuvant. The birds were immunized with the vaccine candidates together with 1×107 or 1×108 colony-forming units of the LTB strain and were subsequently challenged at 3 weeks of age. The 1×107 colony-forming units of the LTB strain showed a greater adjuvant effect with increased levels of serum IgG, intestinal IgA and a potent lymphocyte proliferation response, and yielded higher protection against challenge. Overall, the LTB strain increased the efficacy of the Salmonella -delivered APEC vaccine, indicating that vaccination for APEC along with the LTB strain appears to increase the efficacy for protection against colibacillosis in broiler chickens.

Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies. METHODOLOGY: Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice. PRINCIPAL FINDINGS: Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC. CONCLUSIONS: Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.

Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development.

Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.

Fimbrial subunit protein FaeG expressed in transgenic tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes.

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.

NF-kappaB p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation.

BACKGROUND: Transcription factors have distinct functions in regulating immune responses. During Escherichia coli pneumonia, deficiency of NF-kappaB p50 increases gene expression and neutrophil recruitment, suggesting that p50 normally limits these innate immune responses. p50-deficient mice were used to determine how p50 regulates responses to a simpler, non-viable bacterial stimulus in the lungs, E. coli lipopolysaccharide (LPS). RESULTS: In contrast to previous results with living E. coli, neutrophil accumulation elicited by E. coli LPS in the lungs was decreased by p50 deficiency, to approximately 30% of wild type levels. Heat-killed E. coli induced neutrophil accumulation which was not decreased by p50 deficiency, demonstrating that bacterial growth and metabolism were not responsible for the different responses to bacteria and LPS. p50 deficiency increased the LPS-induced expression of kappaB-regulated genes essential to neutrophil recruitment, including KC, MIP-2, ICAM-1, and TNF-alpha suggesting that p50 normally limited this gene expression and that decreased neutrophil recruitment did not result from insufficient expression of these genes. Neutrophils were responsive to the chemokine KC in the peripheral blood of p50-deficient mice with or without LPS-induced pulmonary inflammation. Interleukin-6 (IL-6), previously demonstrated to decrease LPS-induced neutrophil recruitment in the lungs, was increased by p50 deficiency, but LPS-induced neutrophil recruitment was decreased by p50 deficiency even in IL-6 deficient mice. CONCLUSION: p50 makes essential contributions to neutrophil accumulation elicited by LPS in the lungs. This p50-dependent pathway for neutrophil accumulation can be overcome by bacterial products other than LPS and does not require IL-6.

Coproantibodies to the enteropathogenic Escherichia coli vaccine candidates BfpA and EspB in breastfed and artificially fed children.

A diagnostic test was developed to discriminate active from past enteropathogenic Escherichia coli (EPEC) infection, which uses the affinity-purified recombinant proteins BfpA (bundle-forming pilus (BFP) structural repeating subunit A) and EspB (pore-forming secreted protein B) as reliable markers of virulence to detect antigen-specific coproantibodies by immunoblot analysis, and verification of active typical EPEC infection by gene-specific (bfpA and espB) PCR amplification using DNA extracted directly from specimens and/or culture-enriched preparations. To begin addressing the potential protective role of anti-EPEC antibodies at early age, the prevalence of IgA coproantibodies to these antigens was determined in either breastfed or artificially fed children <2 years of age hospitalized for watery diarrhea.