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Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

ABSTRACT: Background: Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea.

Methods: An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute fast at doses of 109 or 1010 cfu. A second cohort included naïve and rechallenged subjects who had moderate-severe diarrhea and were given the target regimen. Immune responses to important ETEC antigens were assessed.

Results: Among subjects receiving 108 cfu of B7A, overnight fast, or 109 cfu, 90-minute fast, 42.9% (3/7) had moderate-severe diarrhea. Higher attack rates (71.4%; 5/7) occurred in subjects receiving 109 cfu, overnight fast, or 1010 cfu, 90-minute fast. Upon rechallenge with 109 cfu of B7A, overnight fast, 5/11 (45.5%) had moderate-severe diarrhea; the attack rate among concurrently challenge naïve subjects was 57.9% (11/19). Anti-CS6, O148 LPS and LT responses were modest across all groups.

Conclusions: An overnight fast enabled a reduction in the B7A inoculum dose; however, the attack rate was inconsistent and protection upon rechallenge was minimal.

SUBMITTER: Talaat KR

PROVIDER: S-EPMC7710093 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

Talaat Kawsar R KR Porter Chad K CK Jaep Kayla M KM Duplessis Christopher A CA Gutierrez Ramiro L RL Maciel Milton M Adjoodani Brittany B Feijoo Brittany B Chakraborty Subhra S Brubaker Jessica J Trop Stefanie A SA Riddle Mark S MS Joseph Sabrina S SS Bourgeois A Louis AL Prouty Michael G MG

PloS one 20201202 12

<h4>Background</h4>Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea.<h4>Methods</h4>An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute ...[more]

PMID: 33264302

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Project description:IntroductionEnterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)].MethodsThis was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT.ResultsAcross all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays.ConclusionThis is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.

Project description:UnlabelledCS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure.ImportanceUnlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis.

Dataset Information

Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

Publications

Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

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