This report describes the first evaluation in humans of a trivalent, live attenuated ETEC vaccine. ACE527 was developed with the express purpose of overcoming the dual hurdles of inducing immune responses with sufficient magnitude and breadth while maintaining an acceptable reactogenicity profile (45
). In this initial phase I study, ACE527 was safe and well tolerated. Ingestion of ACE527 was not associated with any immediate postvaccination reactions, and serious adverse events were not encountered. There were no unexpected adverse events, on the basis of prior experience with similarly attenuated ETEC strains (10
). Nonserious adverse events occurred with similar frequencies across study groups, except for gastrointestinal symptoms. There was an observed increase in the frequency of gastrointestinal adverse events in the ACE527 high-dose group, but nearly all of these events were mild in severity and all were self-limited. Flatulence was the most common gastrointestinal symptom in this group, reported by nearly two-thirds of high-dose ACE527 recipients. This study therefore extends previous observations with precursor and similarly attenuated ETEC strains and confirms their safety and tolerability up to doses of 1011
). The most significant AEs which were seen (loose stools and nausea) were of a similar frequency and intensity seen in subjects administered equivalent doses of whole-cell killed ETEC vaccines (8
ACE527 was immunogenic following two immunizations given 3 weeks apart, inducing strong immune responses to both toxin and colonization factor antigens at doses of 1010
CFU and 1011
CFU and with dose-dependent increases in the magnitude of anti-LTB and the frequency of anti-CF responses being detected. The predominant systemic immune response was against LTB. A clear dose-response was seen in serum anti-LTB responses, with 10/12 recipients of the higher dose developing at least 2.5-fold rises in IgG. Of these responders, 7 seroconverted after the first dose of vaccine and an additional 3 subjects did so after the second dose. There was no significant further rise in geometric mean titer in the subjects who seroconverted after the first dose after they received the second dose. Mucosal anti-LTB IgA ALS responses were robust, reflecting the strategic design of the three vaccine strains. LTB expression in the three constructs is a mixture of cytoplasmic and periplasmic; there are no specific signal sequences provided to attempt to secrete the product or attach it to the external surface of the cell. Instead, the high level of constitutive expression seen in strains ACAM2022 and ACAM2027 (in which expression is driven by the Ptac
promoter) and the inducible expression in ACAM2025 (where the native LT promoter is used) are relied upon to deliver a dose of antigen sufficient to be immunogenic (41
). This is clearly a successful approach, as 100% of subjects mounted a positive mucosal response to LTB at both dose levels of ACE527, in contrast to the low antibody secreting cells (ASC) response to CT seen following vaccination with Peru15, the live cholera vaccine expressing CTB (29
). This observation suggests that these ETEC vaccine strains may have the potential to also serve as vectors for the expression of additional antigens from other enteric pathogens. The second dose of vaccine increased both the magnitude and breadth of the immune responses generated, supporting the value of a two-dose regimen in travelers; the potential for further increasing the immunogenicity with a third dose should be explored in future studies in children in countries where ETEC is endemic and where an ETEC vaccine is likely to be given according to the routine Expanded Program on Immunization (EPI) schedule.
The dose-response observed here is consistent with the higher responses to a 1010
-CFU dose than to a 109
-CFU dose reported previously with similar strains (10
). There was no obvious relationship between the frequency of immune responses and the number of days on which the vaccine was shed or the magnitude of that shedding in subjects who received the same dose level of ACE527. A similar lack of correlation was seen in phase I studies with a live attenuated cholera vaccine (34
The ability of this vaccine to induce strong anti-LTB and anti-colonization factor mucosal immune responses () is particularly encouraging since animal model, human challenge, and field trial data all indicated that both responses can play an important role in protection against ETEC (1
). The induction of significant anti-LTB, anti-CFA/I, anti-CS3, and anti-CS6 IgA ALS mucosal immune responses in most vaccinees suggests that the vaccine may be capable of inducing local intestinal antibody responses against most of the ETEC strains that may be encountered by travelers or infants and young children living in areas where ETEC is endemic. Although serum antibody responses, particularly to colonization factor antigens, tended to be less frequent than mucosal responses to the same vaccine components (), it was also encouraging that ACE527 induced anti-LTB, anti-CFA/I, and anti-CS6 serum responses that are in the same range as those observed among subjects experimentally infected with wild-type ETEC strains in human challenge studies. Seroconversion of IgA and IgG CFA/I-specific antibodies occurred in 43% and 23% of subjects, respectively, fed H10407 in a study performed by some of the present authors in a similar population and using the identical assay methods (14
); the rates were 27% and 9%, respectively, at the high dose in the present study. In an independent study, the rates of IgA and IgG seroconversion to CS6 in subjects fed the virulent strain B7A were 31% and 25%, respectively (9
), compared to 33% and 9%, respectively, at the high dose in the present study. This study is the first time that an oral cellular ETEC vaccine has been shown to induce both mucosal and serum anti-CS6 responses. Seroconversion to CS3 was not detected in subjects, even though 55% and 50% of vaccinees in the high- and low-vaccine-dose groups, respectively, mounted ALS responses to this antigen. However, baseline serum antibody titers to this antigen tended to be higher than those to CFA/I and CS6, making it more difficult to demonstrate a significant raise following vaccination (data not shown), as previously reported in a similar study population (21
All three vaccine strains were shed in comparable amounts and with comparable durations. The quantitative level of excretion was evaluated 3 days after the first dose and 2 days after the second, with higher counts observed in samples from the latter evaluation, suggesting that excretion is of a short duration, with a peak occurring in the first 2 days after vaccination. The shedding levels are approximately 10-fold higher in the second cohort, in proportion to the higher dose that they received. The peak level observed, 2.2 × 106
CFU of ACE527 per gram of stool, corresponds to 1/50,000 of the administered dose of vaccine per gram of fecal material, reducing the risk of transmission of an effective dose of vaccine to an unintended recipient to essentially zero. This level of shedding of the attenuated vaccine is about 1% of that observed for virulent ETEC strains, such as H10407 (13
) and E24377A (20
). At the higher dose level, the median time to negative stool cultures was less than 7 days after either dose, and the longest time after a vaccine dose where a positive culture was obtained was 21 days. These results are broadly in line with those seen with other live bacterial vaccines, e.g., the cholera vaccine Peru15, where 53% and 13% of vaccinees were excreting vaccine at 7 and 10 days after dosing, respectively (34
), and a newer cholera vaccine candidate, vaccine 638, shown to be shed at a peak of 1.8 × 104
CFU/g, or 1/110,000 of an effective dose per gram (44
). All three strains in ACE527 demonstrate a sufficient duration of colonization for effective antigen expression and induction of immune responses but adequate attenuation to preclude persistent shedding. This correlates with the observation that immune responses to the key antigens expressed on all three strains (CFA/I, CS3, CS6) were seen in the majority of subjects.
Live attenuated vaccines against a number of enteric bacterial diseases have been successfully developed (Vivotif for typhoid fever and Orochol for cholera), with other candidates being in various stages of clinical development (17
). Previously, attenuated ETEC strains expressing different colonization factors have been produced and tested in phase I trials at maximum doses of 2 × 1010
). However, each of these previous studies used single strains of attenuated ETEC, none of which expressed an antigen capable of inducing antitoxin immunity. Since LT is expressed by the majority of ETEC strains and has been shown to be both immunogenic and protective as a stand-alone vaccine antigen, an effective vaccine will be required to induce immune responses to LT (or the nontoxic antigenic B subunit). However, the frequency of strains which express ST either alone or alongside LT makes it unlikely that this will be sufficient to confer broad protection. To protect against these strains, it will also be necessary to induce effective responses to the most prevalent colonization factor antigens, particularly CFA/I, CFA/II, and CFA/IV.
ACE527 shows promise as a well-tolerated live attenuated vaccine that could provide protection against a broad spectrum of ETEC strains. Thus, this vaccine has potential as a public health intervention for both travelers and children at risk for ETEC diarrhea and warrants further development and clinical evaluation. The strong systemic and mucosal antitoxin responses coupled with mucosal responses against principal colonization factor antigens and the positive safety findings in this study support further clinical testing of ACE527 at the 1011-CFU dose in a phase II proof-of-concept vaccination and challenge trial.