scholarly journals Attenuated Shigella flexneri 2a Vaccine Strain CVD 1204 Expressing Colonization Factor Antigen I and Mutant Heat-Labile Enterotoxin of Enterotoxigenic Escherichia coli

2000 ◽  
Vol 68 (9) ◽  
pp. 4884-4892 ◽  
Author(s):  
Hilary Koprowski ◽  
Myron M. Levine ◽  
Richard J. Anderson ◽  
Genevieve Losonsky ◽  
Mariagrazia Pizza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Strain ◽  
Guinea Pigs ◽  
Oral Vaccine ◽  
Shigella Flexneri ◽  
Surface Expression ◽  
Enterotoxigenic Escherichia Coli ◽  
Secretory Iga ◽  
Colonization Factor ◽  
Heat Labile

ABSTRACT A multivalent live oral vaccine against both Shigellaspp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution—serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM1 binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain.

scholarly journals Transcutaneous Immunization Using Colonization Factor and Heat-Labile Enterotoxin Induces Correlates of Protective Immunity for Enterotoxigenic Escherichia coli

2002 ◽  
Vol 70 (3) ◽  
pp. 1056-1068 ◽  
Author(s):  
Jianmei Yu ◽  
Frederick Cassels ◽  
Tanya Scharton-Kersten ◽  
Scott A. Hammond ◽  
Antoinette Hartman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protective Immunity ◽  
Diarrheal Disease ◽  
Surface Protein ◽  
Enterotoxigenic Escherichia Coli ◽  
Vaccine Antigen ◽  
Secretory Iga ◽  
Colonization Factor ◽  
Heat Labile ◽  
Transcutaneous Immunization

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) diarrheal disease is a worldwide problem that may be addressed by transcutaneous delivery of a vaccine. In several human settings, protective immunity has been associated with immune responses to E. coli colonization factors and to the heat-labile toxin that induces the diarrhea. In this set of animal studies, transcutaneous immunization (TCI) using recombinant colonization factor CS6 and cholera toxin (CT) or heat-labile enterotoxin (LT) as the adjuvant induced immunoglobulin G (IgG) and IgA anti-CS6 responses in sera and stools and antibody responses that recognized CS6 antigen in its native configuration. The antitoxin immunity induced by TCI was also shown to protect against enteric toxin challenge. Although immunization with LT via the skin induced mucosal secretory IgA responses to LT, protection could also be achieved by intravenous injection of the immune sera. Finally, a malaria vaccine antigen, merzoite surface protein 142 administered with CT as the adjuvant, induced both merzoite surface protein antibodies and T-cell responses while conferring protective antitoxin immunity, suggesting that both antiparasitic activity and antidiarrheal activity can be obtained with a single vaccine formulation. Overall, our results demonstrate that relevant colonization factor and antitoxin immunity can be induced by TCI and suggest that an ETEC traveler's diarrhea vaccine could be delivered by using a patch.

scholarly journals Importance of Heat-Labile Enterotoxin in Colonization of the Adult Mouse Small Intestine by Human Enterotoxigenic Escherichia coli Strains

2006 ◽  
Vol 74 (2) ◽  
pp. 869-875 ◽  
Author(s):  
Kenneth P. Allen ◽  
Mildred M. Randolph ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Small Intestine ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Small Animal ◽  
Enterotoxigenic Escherichia Coli ◽  
Colonization Factor ◽  
Heat Stable ◽  
Heat Labile ◽  
Colonization Factor Antigen I

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease and infant mortality in developing countries. Studies of ETEC pathogenesis relevant to vaccine development have been greatly hampered by the lack of a suitable small-animal model of infection with human ETEC strains. Here, we demonstrate that adult immunocompetent outbred mice can be effectively colonized with the prototypical human ETEC H10407 strain (colonization factor antigen I; heat-labile and heat-stable enterotoxin positive) and that production of heat-labile holotoxin provides a significant advantage in colonization of the small intestine in this model.

scholarly journals Construction and Characterization of Bivalent Shigella flexneri 2a Vaccine Strains SC608(pCFAI) and SC608(pCFAI/LTB) That Express Antigens from Enterotoxigenic Escherichia coli

2005 ◽  
Vol 73 (1) ◽  
pp. 258-267 ◽  
Author(s):  
Ryan T. Ranallo ◽  
C. Piyumi Fonseka ◽  
Fred Cassels ◽  
Jay Srinivasan ◽  
Malabi M. Venkatesan
Keyword(s):  
Escherichia Coli ◽  
Vaccine Strain ◽  
Shigella Flexneri ◽  
Immunoblot Analysis ◽  
Enterotoxigenic Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Live Attenuated Vaccine ◽  
Fimbrial Subunit ◽  
Semialdehyde Dehydrogenase ◽  
Structural Subunit

ABSTRACT An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens. SC608 is an aspartate semialdehyde dehydrogenase (asd) derivative of SC602 (icsA iuc), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers. When administered orally at a single 104 (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis. Using asd-based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E. coli B subunit of heat-labile enterotoxin (LTB). The expression of each heterologous protein in SC608 was verified by immunoblot analysis. Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay. After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis. All immunized animals were subsequently protected against a challenge with wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test. Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively. These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities. These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.

scholarly journals Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies

2009 ◽  
Vol 78 (1) ◽  
pp. 316-325 ◽  
Author(s):  
Weiping Zhang ◽  
Chengxian Zhang ◽  
David H. Francis ◽  
Ying Fang ◽  
David Knudsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Rabbit Antiserum ◽  
Full Length ◽  
Enterotoxigenic Escherichia Coli ◽  
Farm Animals ◽  
Toxic Activity ◽  
Colonization Factor ◽  
Heat Stable ◽  
Heat Labile

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa11(N→K), STa12(P→F), and STa13(A→Q)] and used the full-length pLT192 as an adjuvant to carry the pSTa toxoid for pLT192:pSTa-toxoid fusion antigens. Rabbits immunized with pLT192:pSTa12 or pLT192:pSTa13 fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT192:pSTa13 fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

scholarly journals Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin–Heat-Labile Toxin Chimera

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Aisling O'Dowd ◽  
Milton Maciel ◽  
Steven T. Poole ◽  
Michael G. Jobling ◽  
Julianne E. Rollenhagen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protective Efficacy ◽  
Antibody Responses ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
B Subunit ◽  
Colonization Factor ◽  
Heat Labile ◽  
Intestinal Adhesion ◽  
Colonization Factors

ABSTRACT Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

scholarly journals Identification of a Gene within a Pathogenicity Island of Enterotoxigenic Escherichia coli H10407 Required for Maximal Secretion of the Heat-Labile Enterotoxin

2000 ◽  
Vol 68 (5) ◽  
pp. 2766-2774 ◽  
Author(s):  
James M. Fleckenstein ◽  
Luther E. Lindler ◽  
Eric A. Elsinghorst ◽  
James B. Dale
Keyword(s):  
Escherichia Coli ◽  
Pathogenicity Island ◽  
Open Reading Frames ◽  
Enterotoxigenic Escherichia Coli ◽  
Loop Model ◽  
Ileal Loop ◽  
E Coli ◽  
Link Type ◽  
Colonization Factor ◽  
Heat Labile

ABSTRACT Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETECH10407 , is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.

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