Generation and characterization of a live attenuated enterotoxigenic Escherichia coli combination vaccine expressing six colonization factors and heat-labile toxin subunit B.

Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, double-blind placebo-controlled phase II challenge trial, both in healthy adult volunteers.

Large duplications at reciprocal translocation breakpoints that might be the counterpart of large deletions and could arise from stalled replication bubbles.

Reciprocal chromosome translocations are often not exactly reciprocal. Most familiar are deletions at the breakpoints, up to megabases in extent. We describe here the opposite phenomenon-duplication of tens or hundreds of kilobases at the breakpoint junction, so that the same sequence is present on both products of a translocation. When the products of the translocation are mapped on the genome, they overlap. We report several of these "overlapping-breakpoint" duplications in breast cancer cell lines HCC1187, HCC1806, and DU4475. These lines also had deletions and essentially balanced translocations. In HCC1187 and HCC1806, we identified five cases of duplication ranging between 46 kb and 200 kb, with the partner chromosome showing deletions between 29 bp and 31 Mb. DU4475 had a duplication of at least 200 kb. Breakpoints were mapped using array painting, i.e., hybridization of chromosomes isolated by flow cytometry to custom oligonucleotide microarrays. Duplications were verified by fluorescent in situ hybridization (FISH), PCR on isolated chromosomes, and cloning of breakpoints. We propose that these duplications are the counterpart of deletions and that they are produced at a replication bubble, comprising two replication forks with the duplicated sequence in between. Both copies of the duplicated sequence would go to one daughter cell, on different products of the translocation, while the other daughter cell would show deletion. These duplications may have been overlooked because they may be missed by FISH and array-CGH and may be interpreted as insertions by paired-end sequencing. Such duplications may therefore be quite frequent.

Construction and phase I clinical evaluation of the safety and immunogenicity of a candidate enterotoxigenic Escherichia coli vaccine strain expressing colonization factor antigen CFA/I.

Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 x 10(9) CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.