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 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.

FIG. 1.

TEER responses of CaCo-2 cells to ST, LT, and culture supernatants. Electrical resistance is measured in ohms and plotted as a percentage of the level at time zero. (A) TEER responses to ST and LT. Open triangles, LB broth; open squares, LB broth supplemented with ST at 100 ng/ml; open circles, LB broth supplemented with LT at 100 ng/ml. (B) TEER responses to WS-1858B and ACAM2010. Open triangles, LB broth; filled squares, WS-1858B culture supernatants; filled circles, ACAM2010 culture supernatants. Each experiment was performed in triplicate, and the mean values were plotted, with error bars equal to the standard deviation.

FIG. 2.

Southern hybridization analysis of covalently closed circular plasmid DNA from ETEC strain WS-1858B and its derivatives. A. Plasmid DNA was electrophoresed through 0.6% agarose and stained with SYBR-Gold (Cambridge Bioscience). The position of large-molecular-size cccDNA standards is indicated on the left of the figure, and derived strains are indicated below each lane. B and C. Southern hybridization results obtained using cfa (B) and estA (C) gene-specific probes are shown. Results obtained using the EAST1 gene probe gave results identical to those obtained with estA. Each strain possessed small-molecular-size plasmids of about 5.1 and 4.2 kb, but only that portion of the gel which includes the large-molecular-size plasmids is shown.

FIG. 3.

Shedding of vaccine strains following ingestion of one or two doses of fresh or frozen ACAM2010. Stool samples obtained from subjects were cultured for the presence of ACAM2010. Graph A shows the number of subjects at each time point following vaccination from which the vaccine strain was recovered: open bars, one dose of fresh vaccine; filled bars, one dose of frozen vaccine; hatched bars, two doses of fresh vaccine; diagonally hatched bars, two doses of frozen vaccine. Graph B shows the levels of shedding in CFU per gram of stool estimated from samples taken 3 days following ingestion of a first or second dose of ACAM2010. Negative stool samples are plotted as 2 CFU. Horizontal bars indicate the mean response in each group.

FIG. 4.

IgA CFA/I-specific ASC responses in recipients of one or two doses of ACAM2010. Shown is a dot plot showing the maximal response seen at day 7 or day 10 in individual subjects following ingestion of one or two doses of ACAM2010 at the highest dose level (third cohort in phase A and all subjects in phase B). Responses are plotted after subtraction of the response on the day of the appropriate vaccination; zero or negative responses are plotted as 0.2. Horizontal bars indicate the mean response in each group.