Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

ABSTRACTA coexpression strategy inSaccharomyces cerevisiaeusing episomal and integrative vectors for theEscherichia coliheat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced byActinobacillus pleuropneumoniaecoupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection withA. pleuropneumoniaewas also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

Download Full-text

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00016-14 ◽

2014 ◽

Vol 21 (6) ◽

pp. 799-807 ◽

Cited By ~ 28

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.

Download Full-text

Expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic tomato (Solanum lycopersicum L.) fruit

Czech Journal of Genetics and Plant Breeding ◽

10.17221/77/2013-cjgpb ◽

2014 ◽

Vol 50 (No. 1) ◽

pp. 26-31 ◽

Cited By ~ 2

Author(s):

N.H. Loc ◽

D.T. Long ◽

T.-G. Kim ◽

M.-S. Yang

Keyword(s):

Escherichia Coli ◽

Solanum Lycopersicum ◽

Blot Hybridization ◽

Transgenic Tomato ◽

Enzyme Linked Immunosorbent Assay ◽

Potential Candidate ◽

B Subunit ◽

Solanum Lycopersicum L ◽

Heat Labile ◽

Enterotoxin B

We report a feasibility study for expressing the LTB protein (Escherichia coli heat-labile enterotoxin B subunit) via Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.). We produced five regenerated plants obtained on the selection medium supplemented with an antibiotic. Stable integrations of the LTB gene into the genome of these plants were confirmed by Southern blot hybridization. Western blot analysis showed that only two of the five T<sub>0 </sub>transgenic tomato plants expressed the pentameric LTB protein in the fruits. An enzyme-linked immunosorbent assay indicated that these two plants synthesized the LTB protein bound specifically to GM1 ganglioside, suggesting that the LTB subunits formed active pentamers. The LTB protein produced in tomatoes can be a potential candidate for inexpensive, safe, and effective plant-based vaccines.

Download Full-text

Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

Plant Molecular Biology Reporter ◽

10.1007/s11105-009-0114-3 ◽

2009 ◽

Vol 27 (3) ◽

pp. 388-399 ◽

Cited By ~ 12

Author(s):

Joon-Soo Sim ◽

Hyo-Kyung Pak ◽

Dong-Sub Kim ◽

Seung-Bum Lee ◽

Yong-Hwan Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Neutralizing Epitope ◽

B Subunit ◽

Heat Labile ◽

Enterotoxin B

Download Full-text

Intradermal or Sublingual Delivery and Heat-Labile Enterotoxin Proteins Shape Immunologic Responses to a CFA/I Fimbria-Derived Subunit Antigen Vaccine against Enterotoxigenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.00460-19 ◽

2019 ◽

Vol 87 (11) ◽

Cited By ~ 2

Author(s):

Milton Maciel ◽

David Bauer ◽

Robin L. Baudier ◽

Jacob Bitoun ◽

John D. Clements ◽

...

Keyword(s):

Escherichia Coli ◽

Antibody Responses ◽

Enterotoxigenic Escherichia Coli ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Agglutination ◽

Local Skin ◽

Content Type ◽

B Subunit ◽

Heat Labile ◽

Functional Antibodies

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 μg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.

Download Full-text

Attenuated Salmonella Gallinarum secreting an Escherichia coli heat-labile enterotoxin B subunit protein as an adjuvant for oral vaccination against fowl typhoid

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2012.09.006 ◽

2012 ◽

Vol 150 (3-4) ◽

pp. 149-160 ◽

Cited By ~ 9

Author(s):

Byung Woo Jeon ◽

Chetan V. Jawale ◽

Seung Hwan Kim ◽

John Hwa Lee

Keyword(s):

Escherichia Coli ◽

Oral Vaccination ◽

Salmonella Gallinarum ◽

Subunit Protein ◽

B Subunit ◽

Heat Labile ◽

Fowl Typhoid ◽

Enterotoxin B

Download Full-text

Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic Peperomia pellucida

Protein Expression and Purification ◽

10.1016/j.pep.2010.02.010 ◽

2010 ◽

Vol 72 (1) ◽

pp. 82-86 ◽

Cited By ~ 11

Author(s):

Nguyen Hoang Loc ◽

Nguyen Hoang Bach ◽

Tae-Geum Kim ◽

Moon-Sik Yang

Keyword(s):

Escherichia Coli ◽

Tissue Culture ◽

B Subunit ◽

Heat Labile ◽

Enterotoxin B

Download Full-text

Escherichia coli Heat-Labile Enterotoxin B Subunit Is a More Potent Mucosal Adjuvant than Its Closely Related Homologue, the B Subunit of Cholera Toxin

Infection and Immunity ◽

10.1128/iai.69.5.3476-3482.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3476-3482 ◽

Cited By ~ 57

Author(s):

Douglas G. Millar ◽

Timothy R. Hirst ◽

Denis P. Snider

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Lymphocyte Proliferation ◽

Vaccine Adjuvants ◽

B Subunit ◽

Heat Labile ◽

Antibody Titers ◽

Enterotoxin B ◽

Draining Lymph Nodes ◽

Stimulation Of

ABSTRACT Although cholera toxin (Ctx) and Escherichia coliheat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

Download Full-text