Strategies to overexpress enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates |
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Authors: | Email author" target="_blank">Joshua?TobiasEmail author Ann-Mari?Svennerholm |
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Institution: | (1) Gothenburg University Vaccine Research Institute (GUVAX) and Department of Microbiology and Immunology, The Sahlgrenska Academy, University of Gothenburg, PO Box 435, Gothenburg, 40530, Sweden |
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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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