| 1. |
- Borde, Annika, 1979, et al.
(författare)
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Preparation and preclinical evaluation of a freeze-dried formulation of a novel combined multivalent whole-cell/B-subunit oral vaccine against enterotoxigenic Escherichia coli diarrhea
- 2016
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Ingår i: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411 .- 1873-3441. ; 108, s. 18-24
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Tidskriftsartikel (refereegranskat)abstract
- A promising liquid killed multivalent whole-cell plus enterotoxin B-subunit oral vaccine against enterotoxigenic Escherichia coli (ETEC), the primary cause of diarrhea among children in low-income countries and travelers to these areas, has recently been developed and tested in preclinical and phase-I and phase-II clinical studies. The vaccine contains killed E. coli bacteria over-expressing the main ETEC colonization factors (CFs) CFA/I, CS3, C5 and C6, and a recombinant enterotoxin B subunit protein (LCTBA) given together with a recently developed enterotoxin-derived adjuvant, dmLT. A dry-powder vaccine formulation should be advantageous especially for use in low-income countries. Here we describe a method to produce a dry-powder formulation by freeze-drying of the vaccine using inulin as stabilizer. Although not completely preventing aggregation of bacteria during freeze-drying, the stabilizer provided both improved overall bacterial morphology and almost complete recovery of the CF and B subunit antigens. Most importantly, oral-intragastric immunization of mice with the freeze-dried vaccine together with dmLT adjuvant elicited strong intestinal mucosal and serum antibody responses against all vaccine antigens, which were comparable to those achieved with the liquid vaccine. Our results indicate the feasibility to use freeze-drying with inulin as stabilizer for preparing a dry-powder formulation of the novel ETEC vaccine with retained oral-mucosal immunogenicity compared to the liquid formulation. © 2016 Elsevier B.V.
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| 2. |
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| 3. |
- Davitt, C. J. H., et al.
(författare)
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A novel adjuvanted capsule based strategy for oral vaccination against infectious diarrhoeal pathogens
- 2016
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659. ; 233, s. 162-173
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Tidskriftsartikel (refereegranskat)abstract
- Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill (R) (SmPill (R)) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill (R) that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill (R) formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.
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| 4. |
- Davitt, C. J. H., et al.
(författare)
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Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines
- 2019
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Ingår i: Mucosal Immunology. - : Elsevier BV. - 1933-0219. ; 12, s. 1055-1064
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Tidskriftsartikel (refereegranskat)abstract
- Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3–4 million cases globally with 100,000–150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral ® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill ® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses. © 2019, The Author(s).
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| 5. |
- Holmgren, Jan, 1944, et al.
(författare)
-
Development and preclinical evaluation of safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant
- 2013
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 31:20, s. 2457-2464
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Tidskriftsartikel (refereegranskat)abstract
- A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTB. A), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant.Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell. +. LCTB. A vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell. +. LCTB. A vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.
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| 6. |
- Jansson, Lena, 1979, et al.
(författare)
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Sulfatide recognition by colonization factor antigen CS6 from enterotoxigenic Escherichia coli.
- 2009
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:2
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Tidskriftsartikel (refereegranskat)abstract
- The first step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections is adhesion of the bacterium to the small intestinal epithelium. Adhesion of ETEC is mediated by a number of antigenically distinct colonization factors, and among these, one of the most commonly detected is the non-fimbrial adhesin coli surface antigen 6 (CS6). The potential carbohydrate recognition by CS6 was investigated by binding of recombinant CS6-expressing E. coli and purified CS6 protein to a large number of variant glycosphingolipids separated on thin-layer chromatograms. Thereby, a highly specific binding of the CS6-expressing E. coli, and the purified CS6 protein, to sulfatide (SO(3)-3Galbeta1Cer) was obtained. The binding of the CS6 protein and CS6-expressing bacteria to sulfatide was inhibited by dextran sulfate, but not by dextran, heparin, galactose 4-sulfate or galactose 6-sulfate. When using recombinantly expressed and purified CssA and CssB subunits of the CS6 complex, sulfatide binding was obtained with the CssB subunit, demonstrating that the glycosphingolipid binding capacity of CS6 resides within this subunit. CS6-binding sulfatide was present in the small intestine of species susceptible to CS6-mediated infection, e.g. humans and rabbits, but lacking in species not affected by CS6 ETEC, e.g. mice. The ability of CS6-expressing ETEC to adhere to sulfatide in target small intestinal epithelium may thus contribute to virulence.
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| 7. |
- Jansson, Lena, 1979, et al.
(författare)
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The major subunit, CfaB, of colonization factor antigen i from enterotoxigenic Escherichia coli is a glycosphingolipid binding protein.
- 2006
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Ingår i: Infection and immunity. - 0019-9567. ; 74:6, s. 3488-97
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial adherence to mucosal surfaces is an important virulence trait of pathogenic bacteria. Adhesion of enterotoxigenic Escherichia coli (ETEC) to the intestine is mediated by a number of antigenically distinct colonization factors (CFs). One of the most common CFs is CFA/I. This has a fimbrial structure composed of a major repeating subunit, CfaB, and a single tip subunit, CfaE. The potential carbohydrate recognition by CFA/I was investigated by binding CFA/I-fimbriated bacteria and purified CFA/I fimbriae to a large number of variant glycosphingolipids separated on thin-layer chromatograms. For both fimbriated bacteria and purified fimbriae, specific interactions could be identified with a number of nonacid glycosphingolipids. These included glucosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, the H5 type 2 pentaglycosylceramide, the Lea-5 glycosphingolipid, the Lex-5 glycosphingolipid, and the Ley-6 glycosphingolipid. These glycosphingolipids were also recognized by recombinant E. coli expressing CFA/I in the absence of tip protein CfaE, as well as by purified fimbriae from the same strain. This demonstrates that the glycosphingolipid-binding capacity of CFA/I resides in the major CfaB subunit.
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| 8. |
- Longet, S., et al.
(författare)
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Thermostability of the coating, antigen and immunostimulator in an adjuvanted oral capsule vaccine formulation
- 2017
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Ingår i: International Journal of Pharmaceutics. - : Elsevier BV. - 0378-5173. ; 534:1-2, s. 60-70
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Tidskriftsartikel (refereegranskat)abstract
- Oral vaccines present an attractive alternative to injectable vaccines for enteric diseases due to ease of delivery and the induction of intestinal immunity at the site of infection. However, susceptibility to gastrointestinal proteolysis, limited transepithelial uptake and a lack of clinically acceptable adjuvants present significant challenges. A further challenge to mass vaccination in developing countries is the very expensive requirement to maintain the cold chain. We recently described the effectiveness of a Single Multiple Pill® (SmPill®) adjuvanted capsule approach to enhance the effectiveness of a candidate enterotoxigenic Escherichia coli (ETEC) oral vaccine. Here it was demonstrated that this delivery system maintains the antigenicity of ETEC colonisation factor antigen I (CFA/I) and the immunostimulatory activity of the orally active α-Galactosylceramide (α-GalCer) adjuvant after storage of SmPill® minispheres under room temperature and extreme storage conditions for several months. In addition, the internal structure of the cores of SmPill® minispheres and antigen release features at intestinal pH were found to be preserved under all these conditions. However, changes in the surface morphology of SmPill® minispheres leading to the antigen release at gastric pH were observed after a few weeks of storage under extreme conditions. Those modifications were prevented by the introduction of an Opadry® White film coating layer between the core of SmPill® minispheres and the enteric coating. Under these conditions, protection against antigen release at gastric pH was maintained even under high temperature and humidity conditions. These results support the potential of the SmPill® minisphere approach to maintain the stability of an adjuvanted whole cell killed oral vaccine formulation. © 2017 Elsevier B.V.
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| 9. |
- Lundgren, Anna, 1974, et al.
(författare)
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Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.
- 2013
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 31:8, s. 1163-1170
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB.
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| 10. |
- Mottram, Lynda, et al.
(författare)
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Glyco-engineered cell line and computational docking studies reveals enterotoxigenic Escherichia coli CFA/I fimbriae bind to Lewis a glycans
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported clinical data to suggest that colonization factor I (CFA/I) fimbriae of enterotoxigenic Escherichia coli (ETEC) can bind to Lewis a (Le(a)), a glycan epitope ubiquitous in the small intestinal mucosa of young children (< 2 years of age), and individuals with a genetic mutation of FUT2. To further elucidate the physiological binding properties of this interaction, we engineered Chinese Hamster Ovary (CHO-K1) cells to express Le(a) or Le(b) determinants on both N- and O-glycans. We used our glyco-engineered CHO-K1 cell lines to demonstrate that CfaB, the major subunit of ETEC CFA/I fimbriae, as well as four related ETEC fimbriae, bind more to our CHO-K1 cell-line expressing Le(a), compared to cells carrying Le(b) or the CHO-K1 wild-type glycan phenotype. Furthermore, using in-silico docking analysis, we predict up to three amino acids (Glu(25), Asn(27), Thr(29)) found in the immunoglobulin (Ig)-like groove region of CfaB of CFA/I and related fimbriae, could be important for the preferential and higher affinity binding of CFA/I fimbriae to the potentially structurally flexible Le(a) glycan. These findings may lead to a better molecular understanding of ETEC pathogenesis, aiding in the development of vaccines and/or anti-infection therapeutics.
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| 11. |
- Nicklasson, Matilda, 1978, et al.
(författare)
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Mutations in the periplasmic chaperone leading to loss of surface expression of the colonization factor CS6 in enterotoxigenic Escherichia coli (ETEC) clinical isolates.
- 2008
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Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010. ; 44:3, s. 246-54
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by adhesion to human enterocytes by one or more colonization factors (CFs) and secretion of heat-labile (LT) and/or heat-stable (ST) enterotoxins. Expression of coli surface antigen 6 (CS6) on the bacterial surface, usually associated with ETEC strains that produce ST alone or in combination with LT, is rarely found in strains expressing only LT. However, a number of LT-only strains which are genotypically positive but phenotypically negative for CS6 have been identified. In this study, eight such strains from India and Guinea-Bissau belonging to different clones were analysed. The CS6 operon cssABCD was transcribed but protein analyses suggested that the structural subunits CssA and CssB of CS6 were absent in the periplasm. Most strains contained truncating mutations within the periplasmic chaperone-encoding gene cssC and protein modelling indicated that this severely affected the substrate-binding capacity of the chaperone. A single-nucleotide polymorphism (SNP) (A-->T) in the 5'-untranslated region of cssC distinguished the eight strains from ETEC strains that do express CS6 on the surface and may be a potential marker for ETEC strains containing phenotypically silent cssABCD. The study emphasizes the importance of using both genotypic and phenotypic methods in epidemiological studies of ETEC, e.g. for vaccine development.
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| 12. |
- Sjöling, Åsa, 1968, et al.
(författare)
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Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels
- 2015
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Ingår i: Microbiological Research. - : Elsevier BV. - 0944-5013 .- 1618-0623. ; 172, s. 34-40
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Tidskriftsartikel (refereegranskat)abstract
- Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples.
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| 13. |
- Svennerholm, Ann-Mari, 1947, et al.
(författare)
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Vaccines against enterotoxigenic Escherichia coli.
- 2008
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Ingår i: Expert review of vaccines. - : Informa UK Limited. - 1744-8395 .- 1476-0584. ; 7:6, s. 795-804
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children less than 3 years of age in developing countries and in travelers to these areas. The key pathogenic mechanisms that contribute to the pathogenesis of ETEC are the production of colonization factors (CFs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin. To provide broad-spectrum protection, an ETEC vaccine should, most likely, contain the most prevalent fimbrial antigens, that is, CF antigen I and CS1-CS6, and/or a LT toxoid. Different strategies have been taken to deliver ETEC fimbriae and toxin antigens to the human immune system to elicit strong mucosal, in particular, intestinal immune responses that are considered to be of prime importance for protection against ETEC disease. There has been some promise when testing different ETEC candidate vaccines for protection against diarrhea in adult travelers. However, no ETEC candidate vaccine has been shown to be effective in the most important target group, which is infants and young children in endemic areas. Against this background, intense efforts are in progress to try to improve the immunogenicity of different available candidate vaccines, as well as to develop new types of ETEC vaccines.
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| 14. |
- Tobias, Joshua, 1969, et al.
(författare)
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Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines.
- 2010
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Ingår i: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 87:4, s. 1355-65
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal-mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.
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| 15. |
- Tobias, Joshua, 1969, et al.
(författare)
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Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice.
- 2011
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 29:48, s. 8863-9
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Tidskriftsartikel (refereegranskat)abstract
- Coli surface antigen 6 (CS6) is one of the most prevalent non-fimbrial colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) bacteria, which are the most common cause of diarrhea among infants and children in developing countries. Since immune protection against ETEC is mainly mediated by locally produced IgA antibodies in the gut, much effort is focused on the development of an oral CF-based vaccine. Previous work has described the preparation of candidate E. coli vaccine strains expressing immunogenic amounts of fimbrial CF antigens such as CFA/I and CS2, which are retained after formalin treatment. However, attempts to generate E. coli expressing immunogenic amounts of CS6 and to preserve the immunological activity of the CS6 protein in a killed whole-cell vaccine have failed until now. Here we describe the construction of a recombinant non-toxigenic E. coli strain, with thyA as a non-antibiotic-based selection, which expresses large amounts of CS6 antigen on the bacterial surface, and show that phenol inactivation of the bacteria does not destroy the CS6 antigen properties. Oral immunization of mice with such phenol-killed CS6 over-expressing E. coli bacteria induced strong fecal and intestinal IgA and serum IgG+IgM antibody responses to CS6 that exceeded the responses induced by an ETEC reference strain naturally expressing CS6 and previously used as a vaccine strain. Our data indicate that the described phenol-inactivated non-toxigenic and CS6 over-expressing E. coli strain may be a useful component in an oral ETEC vaccine.
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| 16. |
- Tobias, Joshua, 1969, et al.
(författare)
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Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae.
- 2008
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X. ; 26:6, s. 743-52
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Tidskriftsartikel (refereegranskat)abstract
- To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.
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| 17. |
- Tobias, Joshua, 1969, et al.
(författare)
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Involvement of main diarrheagenic Escherichia coli, with emphasis on enteroaggregative E. coli, in severe non-epidemic pediatric diarrhea in a high-income country
- 2015
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Ingår i: Bmc Infectious Diseases. - : Springer Science and Business Media LLC. - 1471-2334. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Bacterial and viral enteric pathogens are the leading cause of diarrhea in infants and children. We aimed to identify and characterize the main human diarrheagenic E. coli (DEC) in stool samples obtained from children less than 5 years of age, hospitalized for acute gastroenteritis in Israel, and to examine the hypothesis that co-infection with DEC and other enteropathogens is associated with the severity of symptoms. Methods: Stool specimens obtained from 307 patients were tested by multiplex PCR (mPCR) to identify enteroaggregative E. coli (EAEC), enterohemorrhagic (EHEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). Specimens were also examined for the presence of rotavirus by immunochromatography, and of Shigella, Salmonella and Campylobacter by stool culture; clinical information was also obtained. Results: Fifty nine (19%) children tested positive for DEC; EAEC and atypical EPEC were most common, each detected in 27 (46%), followed by ETEC (n = 3; 5%), EHEC and typical EPEC (each in 1 child; 1.5%). Most EAEC isolates were resistant to cephalexin, cefixime, cephalothin and ampicillin, and genotypic characterization of EAEC isolates by O-typing and pulsed-field gel electrophoresis showed possible clonal relatedness among some. The likelihood of having > 10 loose/watery stools on the most severe day of illness was significantly increased among patients with EAEC and rotavirus co-infection compared to children who tested negative for both pathogens: adjusted odds ratio 7.0 (95% CI 1.45-33.71, P = 0.015). Conclusion: DEC was common in this pediatric population, in a high-income country, and mixed EAEC and rotavirus infection was characterized by especially severe diarrhea.
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| 18. |
- Tobias, Joshua, 1969, et al.
(författare)
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Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together, on non-toxigenic E. coli bacteria
- 2010
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 28:43, s. 6977-6984
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I. +. CS2, induced specific serum IgG. +. IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.
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| 19. |
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| 20. |
- Tobias, Joshua, 1969, et al.
(författare)
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Role of different genes in the CS6 operon for surface expression of Enterotoxigenic Escherichia coli colonization factor CS6.
- 2008
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X. ; 26:42, s. 5373-80
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Tidskriftsartikel (refereegranskat)abstract
- Coli surface antigen 6, CS6, is one of the most prevalent colonization factors (CFs) associated with Enterotoxigenic Escherichia coli (ETEC) bacteria, the most common cause of diarrhea among infants and children in developing countries. The CS6 operon encodes two structural subunit proteins, CssA and CssB, a chaperon, CssC, and an usher, CssD. Since little is known about the relationship of the genes and their role in expression and surface assembly of CS6, the operon was cloned into a laboratory E. coli strain, and mutants were constructed by creating deletions in each of the genes. Examination of protein expression by different methods, using monoclonal antibodies with specificities for the individual CS6 structural proteins, suggested that the usher (CssD) was not involved in assembly or surface expression of CS6 whereas deletion of the chaperon (CssC) significantly reduced levels of CssA, but not of CssB. Binding studies with CaCo-2 cells demonstrated that there is a combined effect of CssC and CssD on receptor binding, presumably associated with their activities in assembly and transport of the structural subunits. These findings may have important implications for the construction of strains expressing high levels of CS6 on the surface for eventual use in an ETEC vaccine.
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| 21. |
- Tobias, Joshua, 1969, et al.
(författare)
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Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli
- 2012
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Ingår i: Microbiological Research. - : Elsevier BV. - 0944-5013. ; 167:9, s. 564-570
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Tidskriftsartikel (refereegranskat)abstract
- Establishment of a simple and rapid multiplex PCR system for identification of the main diarrheagenic E. coli categories, including enteroaggregative E. coil. enterotoxigenic E. coil, enteropathogenic E. coil, and enterohemorrhagic E. coil, is described. This two-step multiplex PCR system allows the identification by targeting CVD432, LT, STh, Sip, Eae, Bfp, Stx1, and Stx2. By applying the developed multiplex PCR system, categorization of E. coil isolates isolated from stool samples of infants with diarrhea into the main diarrheagenic E. coil categories is also shown. (C) 2011 Elsevier GmbH. All rights reserved.
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| 22. |
- Tobias, Joshua, 1969, et al.
(författare)
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Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)
- 2016
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC iso-lates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.
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| 23. |
- Tobias, Joshua, 1969, et al.
(författare)
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Strategies to overexpress enterotoxigenic Escherichia coli (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates.
- 2012
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Ingår i: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 93:6, s. 2291-300
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Forskningsöversikt (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveler's diarrhea (TD). Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. The most extensively studied ETEC candidate vaccine is the rCTB-CF ETEC vaccine, containing recombinantly produced cholera B subunit and the most commonly encountered ETEC CFs on the surface of whole inactivated bacteria. Initial clinical trials with this vaccine showed significant immune responses against the key antigens in different age groups in Bangladesh and Egypt and protection against more severe TD in Western travelers. However, when tested in a phase-III trial in Egyptian infants, the protective efficacy of the vaccine was found to be low, indicating the need to improve the immunogenicity of the vaccine, e.g., by increasing the levels of the protective antigens. This review describes different strategies for the construction of recombinant nontoxigenic E. coli and Vibrio cholerae candidate vaccine strains over-expressing higher amounts of ETEC CFs than clinical ETEC isolates selected to produce high levels of the respective CF, e.g., those ETEC strains which have been used in the rCTB-CF ETEC vaccine. Several different expression vectors containing the genes responsible for the expression and assembly of the examined CFs, all downstream of the powerful tac promoter, which could be maintained either with or without antibiotic selection, were constructed. Expression from the tac promoter was under the control of the lacI(q) repressor present on the plasmids. Following induction with isopropyl-β-D-thiogalactopyranoside, candidate vaccine strains over-expressing single CFs, unnatural combinations of two CFs, and also hybrid forms of ETEC CFs were produced. Specific monoclonal antibodies against the major subunits of the examined CF were used to quantify the amount of the surface-expressed CF by a dot-blot assay and inhibition ELISA. Oral immunization with formalin- or phenol-inactivated recombinant bacteria over-expressing the CFs was found to induce significantly higher antibody responses compared to immunization with the previously used vaccine strains. We therefore conclude that our constructs may be useful as candidate strains in an oral whole-cell inactivated CF ETEC vaccine.
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| 24. |
- Tobias, Joshua, 1969, et al.
(författare)
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Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens
- 2017
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Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 105, s. 177-184
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae 01 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V cholerae strain co expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori. (C) 2017 Elsevier Ltd. All rights reserved.
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| 25. |
- von Mentzer, Astrid, 1983, et al.
(författare)
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Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC)
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Although 22 antigenically different colonization factors (CFs) have been identified and characterized in ETEC at least 30% of clinical ETEC isolates lack known CFs. Ninety-four whole genome sequenced "CF negative" isolates were searched for novel CFs using a reverse genetics approach followed by phenotypic analyses. We identified a novel CF, CS30, encoded by a set of seven genes, csmA-G, related to the human CF operon CS18 and the porcine CF operon 987P (F6). CS30 was shown to be thermo-regulated, expressed at 37 degrees C, but not at 20 degrees C, by SDS-page and mass spectrometry analyses as well as electron microscopy imaging. Bacteria expressing CS30 were also shown to bind to differentiated human intestinal Caco-2 cells. The genes encoding CS30 were located on a plasmid (E873p3) together with the genes encoding LT and STp. PCR screening of ETEC isolates revealed that 8.6% (n = 13) of "CF negative" (n = 152) and 19.4% (n = 13) of " CF negative" LT + STp (n = 67) expressing isolates analyzed harbored CS30. Hence, we conclude that CS30 is common among " CF negative" LT + STp isolates and is associated with ETEC that cause diarrhea. RAHAM JM, 1985, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES
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