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

Abstract

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 ؋ 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.

Generation and Characterization of a Live Attenuated Enterotoxigenic Escherichia coli Combination Vaccine Expressing Six Colonization Factors and Heat-Labile Toxin Subunit B

“…The loss of another plasmid of ϳ70 kb during construction of ACAM2007 is also apparent. The loss of ST was also observed to coincide with a large deletion from a large-molecular-size plasmid during construction of previously described vaccine candidate ACAM2010 (31). These results demonstrate the propensity of ETEC plasmids to undergo substantial rearrangements which can be isolated when cultures are placed under strong selective pressure.…”

“…Vaccine strains ACAM2022, ACAM2025, and ACAM2027 all express the LTB subunit and between them express CFA/I, CS1, CS2, CS3, CS5, and CS6. Precursors of ACAM2025 (ACAM2010) and ACAM2027 (ACAM2007 and ACAM2017) have already been demonstrated to be safe and immunogenic in human volunteers (5,31), as has PTL003, which was similarly attenuated (15,32).…”

“…The presence or absence of ST and LT in bacterial culture supernatants was confirmed using transepithelial electrical resistance (TEER) of CaCo-2 cells monitored using chopstick electrodes (World Precision Instruments, Stevenage, United Kingdom) which can distinguish between ST and/or LT (31). The absence of LT expression in toxinnegative strains was also confirmed using a Y1 adrenal cell toxicity assay (6).…”

“…Construction of suicide vectors for deletion of astA, eltAB, estA, aroC, ompC, and ompF. The plasmid constructs used are listed in Table 1, and many have been described previously (31,32). Plasmid pJCB12-⌬estA E incorporates a defined deletion mutation of the ST gene from strain WS-2773E.…”

“…The pJCB12 derivatives used to introduce deletion mutations into aroC, ompC, and ompF have been described previously (31).…”