scholarly journals Preclinical characterization of immunogenicity and efficacy against diarrhea from MecVax, a multivalent enterotoxigenic E. coli vaccine candidate

2021 ◽  
Author(s):  
Hyesuk Seo ◽  
Carolina Garcia ◽  
Xiaosai Ruan ◽  
Qiangde Duan ◽  
David A Sack ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Developing Countries ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Preclinical Study ◽  
Watery Diarrhea ◽  
E Coli ◽  
Heat Stable ◽  
Protective Antibodies

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied toxoid fusion strategy and novel epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and adhesin CFA/I/II/IV MEFA, and demonstrated that proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I, CS1 to CS6) expressed by the ETEC strains causing 60-70% diarrheal cases and moderate-to-severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and CT enterotoxicity. Moreover, MecVax protected against watery diarrhea, and over 70% or 90% any diarrhea from an STa+ or an LT+ ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying MecVax potentially an effective injectable vaccine for ETEC. IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children’s diarrhea and travelers’ diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective anti-adhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing enterotoxicity of LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa+ or LT+ ETEC infection in a pig challenge model, recording protection from antibodies induced by protein-based injectable subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of IM administered protein vaccines for protection against intestinal mucosal infection.

Intradermally administered enterotoxigenic E. coli vaccine candidate MecVax induces functional serum IgG antibodies against seven adhesins (CFA/I, CS1-CS6) and both toxins (STa, LT)

2021 ◽  
Author(s):  
Carolina Y Garcia ◽  
Hyesuk Seo ◽  
David A Sack ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Vaccine Candidate ◽  
Effective Vaccine ◽  
Protein Antigens ◽  
E Coli ◽  
Administration Routes ◽  
Heat Stable ◽  
Functional Antibodies

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading bacterial cause of children’s diarrhea and travelers’ diarrhea. MecVax, a multivalent E. coli vaccine candidate composed of two epitope- and structure-based polyvalent proteins (toxoid fusion 3xSTa N12S -mnLT R192G/L211A and CFA/I/II/IV MEFA), is to induce broad anti-adhesin and antitoxin antibodies against heterogeneous ETEC pathovars. Administered intraperitoneally (IP) or intramuscularly (IM), MecVax was shown to induce antibodies against seven ETEC adhesins (CFA/I, CS1-CS6), which are produced by ETEC pathovars causing over 60% of ETEC-associated diarrheal cases and the moderate-to-severe cases, and both toxins (heat-labile toxin - LT and heat-stable toxin - STa) expressed by all ETEC strains. To further characterize immunogenicity of this protein-based injectable subunit vaccine candidate and to explore other parenteral administration routes for the product, in this study, we intradermally (ID) immunized mice with MecVax and measured antigen-specific antibody responses and further antibody functional activities against the adhesins and toxins targeted by the vaccine. Data showed that mice ID immunized with MecVax developed robust anti-CFA/I, -CS1, -CS2, -CS3, -CS4, -CS5, -CS6, -LT and anti-STa IgG responses. Furthermore, antibodies derived from MecVax via ID route inhibited adherence of ETEC or E. coli strains expressing any of the seven target adhesins (CFA/I, CS1-CS6) and neutralized enterotoxicity of LT and STa toxins. These results confirmed broad immunogenicity of MecVax and suggested that this multivalent ETEC subunit vaccine candidate can be effectively delivered via ID route. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of diarrhea in children living in developing countries and international travelers. Developing an effective vaccine for ETEC diarrhea has been hampered because of challenges of virulence heterogeneity and difficulties of inducing neutralizing antibodies against the key STa toxin. MecVax, a subunit vaccine candidate carrying two polyvalent protein antigens for the first time induces functional antibodies against the most important ETEC adhesins which are associated with a majority of diarrheal cases and the moderate-to-severe cases but also against enterotoxicity of LT and more importantly STa toxin which plays a key role in children’s diarrhea and travelers’ diarrhea, potentially leading to development of a truly effective ETEC vaccine. Data from this study may also indicated that this ETEC subunit vaccine can be administered effectively via ID route, expanding clinical administration options for this vaccine product.

Primary structure determination of Escherichia coli heat-stable enterotoxin of porcine origin

1983 ◽  
Vol 61 (5) ◽  
pp. 287-292 ◽  
Author(s):  
Claude Lazure ◽  
Nabil G. Seidah ◽  
Michel Chrétien ◽  
Réal Lallier ◽  
Serge St-Pierre
Keyword(s):  
Escherichia Coli ◽  
Chemical Characterization ◽  
Coli Strain ◽  
Disulfide Bridges ◽  
E Coli ◽  
Heat Stable ◽  
Porcine Origin ◽  
Quantitative Amino Acid Analysis

The chemical characterization of Escherichia coli heat-stable enterotoxin (ST) is described. The toxin was isolated and purified to homogeneity from the E. coli strain F11 (PI55) of porcine origin. Following quantitative amino acid analysis, the enterotoxin was found to contain 18 amino acids including 6 cysteines, but was devoid of Ser, Val, Met, Ile, Lys, His, and Arg residues. All cysteine residues were found to be involved in disulfide bridges, even though their positions could not be localized. The enterotoxin thus has a molecular weight of 1979 and was shown to be an octadecapeptide with the following sequence: H2N-Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr-COOH. Its relationship to other known enterotoxins is discussed.

scholarly journals Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV

2013 ◽  
Vol 21 (2) ◽  
pp. 243-249 ◽  
Author(s):  
Xiaosai Ruan ◽  
David E. Knudsen ◽  
Katie M. Wollenberg ◽  
David A. Sack ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Bacterial Colonization ◽  
Content Type ◽  
E Coli ◽  
Colonization Factor ◽  
Protective Antibodies ◽  
Factor Antigen ◽  
Fusion Antigen

ABSTRACTDiarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. EnterotoxigenicEscherichia coli(ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC orE. colistrains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence ofE. colistrains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

scholarly journals Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins

Toxins ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 274 ◽  
Author(s):  
Morten Govasli ◽  
Yuleima Diaz ◽  
Ephrem Zegeye ◽  
Christine Darbakk ◽  
Arne Taxt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Candidate ◽  
Enterotoxigenic Escherichia Coli ◽  
Purification And Characterization ◽  
Heat Stable

scholarly journals MacAB Is Involved in the Secretion of Escherichia coli Heat-Stable Enterotoxin II

2008 ◽  
Vol 190 (23) ◽  
pp. 7693-7698 ◽  
Author(s):  
Hiroyasu Yamanaka ◽  
Hidetomo Kobayashi ◽  
Eizo Takahashi ◽  
Keinosuke Okamoto
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bonds ◽  
Watery Diarrhea ◽  
Accessory Proteins ◽  
E Coli ◽  
Extracellular Secretion ◽  
Heat Stable ◽  
Atp Binding Cassette Transporter ◽  
Precursor Proteins ◽  
Peptide Toxin

ABSTRACT The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.

scholarly journals Confronting Challenges to Enterotoxigenic Escherichia coli Vaccine Development

2021 ◽  
Vol 2 ◽  
Author(s):  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Symptomatic Infection ◽  
Middle Income ◽  
E Coli ◽  
Heat Stable ◽  
Structural Details ◽  
Basic Sanitation ◽  
Diarrheal Morbidity

The enterotoxigenic Escherichia coli (ETEC) are a diverse and genetically plastic pathologic variant (pathovar) of E. coli defined by their production of heat-labile (LT) and heat-stable (ST) enterotoxins. These pathogens, which came to recognition more than four decades ago in patients presenting with severe cholera-like diarrhea, are now known to cause hundreds of millions of cases of symptomatic infection annually. Children in low-middle income regions of the world lacking access to clean water and basic sanitation are disproportionately affected by ETEC. In addition to acute diarrheal morbidity, these pathogens remain a significant cause of mortality in children under the age of five years and have also been linked repeatedly to sequelae of childhood malnutrition and growth stunting. Vaccines that could prevent ETEC infections therefore remain a high priority. Despite several decades of effort, a licensed vaccine that protects against the breadth of these pathogens remains an aspirational goal, and the underlying genetic plasticity of E. coli has posed a fundamental challenge to development of a vaccine that can encompass the complete antigenic spectrum of ETEC. Nevertheless, novel strategies that include toxoids, a more complete understanding of ETEC molecular pathogenesis, structural details of target immunogens, and the discovery of more highly conserved antigens essential for virulence should accelerate progress and make a broadly protective vaccine feasible.

scholarly journals Molecular characterization of enterotoxigenic Escherichia coli toxins and colonization factors in children under five years with acute diarrhea attending Kisii Teaching and Referral Hospital, Kenya

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Erick Kipkirui ◽  
Margaret Koech ◽  
Abigael Ombogo ◽  
Ronald Kirera ◽  
Janet Ndonye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acute Diarrhea ◽  
Enterotoxigenic Escherichia Coli ◽  
Medical Attention ◽  
Toxin Gene ◽  
E Coli ◽  
Heat Stable ◽  
Colonization Factors ◽  
Porcine Origin

Abstract Background Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infectious diarrhea in children. There are no licensed vaccines against ETEC. This study aimed at characterizing Escherichia coli for ETEC enterotoxins and colonization factors from children < 5 years with acute diarrhea and had not taken antibiotics prior to seeking medical attention at the hospital. Methods A total of 225 randomly selected archived E. coli strains originally isolated from 225 children with acute diarrhea were cultured. DNA was extracted and screened by multiplex polymerase chain reaction (PCR) for three ETEC toxins. All positives were then screened for 11 colonization factors by PCR. Results Out of 225 E. coli strains tested, 23 (10.2%) were ETEC. Heat-stable toxin (ST) gene was detected in 16 (69.6%). ETEC isolates with heat-stable toxin of human origin (STh) and heat-stable toxin of porcine origin (STp) distributed as 11 (68.8%) and 5 (31.2%) respectively. Heat-labile toxin gene (LT) was detected in 5 (21.7%) of the ETEC isolates. Both ST and LT toxin genes were detected in 2 (8.7%) of the ETEC isolates. CF genes were detected in 14 (60.9%) ETEC strains with a majority having CS6 6 (42.9%) gene followed by a combination of CFA/I + CS21 gene detected in 3 (21.4%). CS14, CS3, CS7 and a combination of CS5 + CS6, CS2 + CS3 genes were detected equally in 1 (7.1%) ETEC isolate each. CFA/I, CS4, CS5, CS2, CS17/19, CS1/PCFO71 and CS21 genes tested were not detected. We did not detect CF genes in 9 (39.1%) ETEC isolates. More CFs were associated with ETEC strains with ST genes. Conclusion ETEC strains with ST genes were the most common and had the most associated CFs. A majority of ETEC strains had CS6 gene. In 9 (39.1%) of the evaluated ETEC isolates, we did not detect an identifiable CF.

scholarly journals Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ (SslE)

2021 ◽  
Vol 12 ◽  
Author(s):  
Saman Riaz ◽  
Hans Steinsland ◽  
Mette Thorsing ◽  
Ann Z. Andersen ◽  
Anders Boysen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Fold Increase ◽  
Antibody Responses ◽  
E Coli ◽  
Flow Cytometric ◽  
Median Proportion ◽  
Iga Response ◽  
Iga Antibody

Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

1991 ◽  
Vol 37 (5) ◽  
pp. 407-410
Author(s):  
Mônica A. M. Vieira ◽  
Beatriz E. C. Guth ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Sensitivity And Specificity ◽  
Dna Probes ◽  
Type I ◽  
Enteropathogenic Escherichia Coli ◽  
Key Words Dna ◽  
E Coli ◽  
Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

Sign in / Sign up

Export Citation Format

Share Document