scholarly journals A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

2013 ◽  
Vol 81 (5) ◽  
pp. 1532-1540 ◽  
Author(s):  
Louise Sjökvist Ottsjö ◽  
Carl-Fredrik Flach ◽  
John Clements ◽  
Jan Holmgren ◽  
Sukanya Raghavan
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Immune Responses ◽  
Double Mutant ◽  
Bacterial Load ◽  
Mucosal Vaccine ◽  
Mucosal Adjuvant ◽  
Content Type ◽  
Heat Labile ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine againstH. pyloriinfection is available to date. In mice, mucosal vaccination withH. pyloriantigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses againstH. pyloriinfection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutantEscherichia coliheat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimentalH. pylorivaccine and compared it to CT in promoting immune responses and protection againstH. pyloriinfection in mice. Immunization via the sublingual or intragastric route withH. pylorilysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhancedin vitroproliferative and cytokine responses from spleen and mesenteric lymph node cells toH. pyloriantigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine againstH. pyloriinfection.

scholarly journals The A Subunit of Escherichia coli Heat-Labile Enterotoxin Functions as a Mucosal Adjuvant and Promotes IgG2a, IgA, and Th17 Responses to Vaccine Antigens

2012 ◽  
Vol 80 (7) ◽  
pp. 2426-2435 ◽  
Author(s):  
Elizabeth B. Norton ◽  
Louise B. Lawson ◽  
Zaid Mahdi ◽  
Lucy C. Freytag ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Fluid Secretion ◽  
Proteolytic Degradation ◽  
Mucosal Adjuvant ◽  
Content Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Camp Induction

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) produces both heat-labile (LT) and heat-stable (ST) enterotoxins and is a major cause of diarrhea in infants in developing countries and in travelers to those regions. In addition to inducing fluid secretion, LT is a powerful mucosal adjuvant capable of promoting immune responses to coadministered antigens. In this study, we examined purified A subunit to further understand the toxicity and adjuvanticity of LT. Purified A subunit was enzymatically active but sensitive to proteolytic degradation and unable to bind gangliosides, and even in the presence of admixed B subunit, it displayed low cyclic AMP (cAMP) induction and no enterotoxicity. Thus, the AB5 structure plays a key role in protecting the A subunit from proteolytic degradation and in delivering the enzymatic signals required for secretion. In contrast, the A subunit alone was capable of activating dendritic cells and enhanced immune responses to multiple antigens following intranasal immunization; therefore, unlike toxicity, LT adjuvanticity is not dependent on the AB5 holotoxin structure or the presence of the B subunit. However, immune responses were maximal when signals were received from both subunits either in an AB5 structure or with A and B admixed. Furthermore, the quality of the immune response (i.e., IgG1/IgG2 balance and mucosal IgA and IL-17 secretion) was determined by the presence of an A subunit, revealing for the first time induction of Th17 responses with the A subunit alone. These results have important implications for understanding ETEC pathogenesis, unraveling immunologic responses induced by LT-based adjuvants, and developing new mucosal vaccines.

scholarly journals A Novel Line Immunoassay Based on Recombinant Virulence Factors Enables Highly Specific and Sensitive Serologic Diagnosis of Helicobacter pylori Infection

2013 ◽  
Vol 20 (11) ◽  
pp. 1703-1710 ◽  
Author(s):  
Luca Formichella ◽  
Laura Romberg ◽  
Christian Bolz ◽  
Michael Vieth ◽  
Michael Geppert ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Responses ◽  
Virulence Factors ◽  
Gastrointestinal Disease ◽  
Individual Risk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Serologic Test ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloricolonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome.H. pylorivirulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to importantH. pylorivirulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed inEscherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) wereH. pylorinegative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), therecomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, therecomLine assay provides a valuable tool for the diagnosis ofH. pyloriinfection.

scholarly journals Helicobacter pylori Peptidoglycan Modifications Confer Lysozyme Resistance and Contribute to Survival in the Host

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Ge Wang ◽  
Leja F. Lo ◽  
Lennart S. Forsberg ◽  
Robert J. Maier
Keyword(s):  
Cell Wall ◽  
Helicobacter Pylori ◽  
Double Mutant ◽  
Lytic Enzyme ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
H Pylori ◽  
Modification Enzyme

ABSTRACTThe prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized aHelicobacter pyloriPG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that apatAstrain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA inH. pyloriPG O-acetylation. The PG modification mutant strains (pgdA,patA, orpgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity toH. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). ThepgdA patAdouble mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from apgdA patAmutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.IMPORTANCEPathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that twoHelicobacter pyloriPG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

scholarly journals Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori

2013 ◽  
Vol 81 (10) ◽  
pp. 3880-3893 ◽  
Author(s):  
Rebecca J. Gorrell ◽  
Odilia L. C. Wijburg ◽  
John S. Pedersen ◽  
Anna K. Walduck ◽  
Terry Kwok ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibody Response ◽  
Bacterial Load ◽  
Secretory Iga ◽  
Mouse Strains ◽  
Wild Type ◽  
Content Type ◽  
Immunoglobulin Receptor ◽  
H Pylori ◽  
Secretory Antibodies

ABSTRACTThe natural immune response toHelicobacter pylorineither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course ofH. pyloriinfection has not been determined. We compared the natural progression ofH. pyloriinfection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces.H. pyloriSS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication ofH. pylorifrom the intestine of wild-type animals by 3 mpi.H. pyloriwas cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load ofH. pyloriin wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress ofH. pyloriinfection, particularly in the duodenum.

scholarly journals Salmonella enterica Serovar Enteritidis Ghosts Carrying the Escherichia coli Heat-Labile Enterotoxin B Subunit Are Capable of Inducing Enhanced Protective Immune Responses

2014 ◽  
Vol 21 (6) ◽  
pp. 799-807 ◽  
Author(s):  
Chetan V. Jawale ◽  
John Hwa Lee
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Salmonella Enterica ◽  
Secretory Iga ◽  
Content Type ◽  
B Subunit ◽  
Heat Labile ◽  
Group A ◽  
Enterotoxin B ◽  
Group B

ABSTRACTTheEscherichia coliheat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant.Salmonella entericaserovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins.S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB inS. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with eitherS. Enteritidis-LTB ghosts orS. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n= 20 for each): group A, nonvaccinated control; group B, immunized withS. Enteritidis-LTB ghosts; and group C, immunized withS. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3+CD4+subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization withS. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization withS. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis.

scholarly journals A Gastric Pathogen Moves Chemotaxis in a New Direction

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Emily Goers Sweeney ◽  
Karen Guillemin
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Chemical Cues ◽  
Swimming Behavior ◽  
Human Pathogen ◽  
Gastric Glands ◽  
Content Type ◽  
Gastric Pathogen ◽  
H Pylori ◽  
Novel Antibiotics

ABSTRACTFor almost 50 years,Escherichia colihas been the model for understanding how bacteria orient their movement in response to chemical cues, but recent studies of chemotaxis in other bacteria have revealed interesting variations from prevailing paradigms. Investigating the human pathogenHelicobacter pylori, Amieva and colleagues [mBio 2(4):e00098-11, 2011] discovered a new chemotaxis regulator, ChePep, which modulates swimming behavior through the canonical histidine-aspartate phosphorelay system. Functionally conserved among the epsilonproteobacteria, ChePep is essential forH. pylorito navigate deep into the stomach’s gastric glands and may be an attractive target for novel antibiotics.

scholarly journals Safety and Immunogenicity of a Single Oral Dose of Recombinant Double Mutant Heat-Labile Toxin Derived from Enterotoxigenic Escherichia coli

2013 ◽  
Vol 20 (11) ◽  
pp. 1764-1770 ◽  
Author(s):  
Samer S. El-Kamary ◽  
Mitchell B. Cohen ◽  
A. Louis Bourgeois ◽  
Lillian Van De Verg ◽  
Nicole Bauers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Dose ◽  
Immune Responses ◽  
Single Oral Dose ◽  
Double Mutant ◽  
Phase 1 ◽  
Enterotoxigenic Escherichia Coli ◽  
Heat Labile ◽  
Serum Igg ◽  
Serum Iga

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a primary cause of traveler's diarrhea for which there is no licensed vaccine. This phase 1 trial determined the safety and immunogenicity of a recombinantly produced double mutant heat-labile enterotoxin (dmLT) of ETEC. It was administered as a single oral dose of dmLT in escalating doses of 5 μg, 25 μg, 50 μg, and 100 μg, followed by a 72-h inpatient observation, outpatient visits at 8, 14, and 28 days, and telephone calls at 2 and 6 months postvaccination. Safety was assessed by frequency of adverse events, and immune responses determined after immunization included dmLT-specific serum IgA and IgG, fecal IgA, antibody-secreting cells (ASC), and antibodies in lymphocyte supernatant (ALS) responses. All doses were well tolerated by the 36 healthy adults enrolled. Immune responses were limited in the 5- and 25-μg dose recipients. The 50-μg dose recipients trended toward stronger responses than the 100-μg dose recipients by serum IgA (67% versus 33%,P= 0.22), serum IgG (58% versus 33%,P= 0.41), and fecal IgA (58% versus 33%,P= 0.41). By day 14 postvaccination, there were significantly more positive responders (≥4-fold increase from baseline) among the 50- versus 100-μg dose recipients for serum IgA (P= 0.036) but not serum IgG (P= 0.21). In conclusion, a single oral dose of dmLT was well tolerated and immunogenic, with immune responses plateauing at the 50-μg dose. (This clinical trial is registered atwww.clinicaltrials.gov, registration number NCT01147445.)

scholarly journals Generation of Helicobacter pylori Ghosts by PhiX Protein E-Mediated Inactivation and Their Evaluation as Vaccine Candidates

2003 ◽  
Vol 71 (1) ◽  
pp. 109-116 ◽  
Author(s):  
Klaus Panthel ◽  
Wolfgang Jechlinger ◽  
Alexander Matis ◽  
Manfred Rohde ◽  
Michael Szostak ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Shuttle Vector ◽  
Bacterial Load ◽  
Gene Cassette ◽  
Resistant Bacteria ◽  
Mucosal Adjuvant ◽  
Vaccine Candidates ◽  
Lysis Gene ◽  
H Pylori ◽  
Cell Envelopes

ABSTRACT Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria to obtain vaccine candidates. We describe here the application of this technology to Helicobacter pylori. The lysis gene cassette was cloned into an Escherichia coli-Helicobacter pylori shuttle vector and introduced into an H. pylori recipient strain by bacterial conjugation. Temperature induction of the lysis gene cassette revealed a quantitative killing of the H. pylori culture without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact H. pylori. Prophylactic oral vaccination experiments using these H. pylori ghosts in the BALB/c mouse model showed a significant reduction of the bacterial load in the ghost group, as measured by a quantitative bacterial reisolation procedure. Ten of 10 and 5 of 10 mice were protected, respectively, without the use of a mucosal adjuvant. Coadministration of ghosts with cholera toxin as mucosal adjuvant resulted in a complete protection of 10 of 10 and 8 of 8 mice against H. pylori challenge, with three animals showing a sterile immunity.

scholarly journals Immunological Response to Parenteral Vaccination with Recombinant Hepatitis B Virus Surface Antigen Virus-Like Particles Expressing Helicobacter pylori KatA Epitopes in a Murine H. pylori Challenge Model

2011 ◽  
Vol 19 (2) ◽  
pp. 268-276 ◽  
Author(s):  
Michael Kotiw ◽  
Megan Johnson ◽  
Manisha Pandey ◽  
Scott Fry ◽  
Stuart L. Hazell ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Bacterial Load ◽  
Igg Antibody ◽  
Virus Surface ◽  
Content Type ◽  
Virus Like Particles ◽  
B Virus ◽  
H Pylori

ABSTRACTVirus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of theHelicobacter pylori katAgene product into HBsAg-S. The HBsAg-S–KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 103) were significantly greater (P< 0.05) than those observed for vaccination with VLP alone (5.2 × 102). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 104and 2.6 × 104, respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P< 0.05). Following challenge of mice withH. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P< 0.05). This is the first report describing the use of VLPs as a delivery vehicle forH. pyloriantigens.

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