The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.

Background Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. Methods We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5•5 × 10¹⁰ cells], quarter [2•5 × 10¹⁰ cells], or eighth [1•25 × 10¹⁰ cells] adult dose), with or without dmLT adjuvant (2•5 µg, 5•0 µg, or 10•0 µg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802.

Of 469 recently isolated Shigella flexneri strains, 452 agglutinated with Shigella flexneri-specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with S. flexneri 2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.Shigellosis is one of the major diarrheal diseases in Bangladesh and several other developing countries and is caused by any one of the four species or groups of Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. Each serogroup contains multiple serotypes, and at least 47 serotypes are currently recognized based on their biochemical and/or lipopolysaccharide characteristics. Based on the structure of the O antigen, a component of the lipopolysaccharide present on the outer membrane of the cell (16), 15 serotypes of S. flexneri are currently recognized. Since S. flexneri serotype 2a was found to be the major endemic serotype in developing countries, the currently available candidate vaccine is directed against S. flexneri 2a (12). It is important to determine the prevalence of various serotypes of S. flexneri in different communities worldwide and to monitor the changes over time.

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.

Highlights The killed oral ETEC vaccine ETVAX ± dmLT adjuvant was safe in Bangladeshi adults. All vaccinees responded to all 5 primary vaccine antigens in ALS specimens. A majority of vaccinees responded to ≥4 antigens in plasma specimens. A sensitive electrochemiluminescence assay was established for small sample volumes. ALS responses measured by electrochemiluminescence and ELISA assays correlated well.

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