This study extends our previous evaluation of the safety, tolerability, and immunogenicity of the three-strain, oral ETEC vaccine, ACE527. In addition, the impact of vaccination on the clinical outcome of experimental challenge with the highly virulent ETEC strain H10407 was evaluated, along with the ability of the vaccine to interfere with colonization by the challenge strain. While the 27% reduction in the incidence of the primary endpoint (moderate to severe diarrhea) was not statistically significant, there were trends toward improved outcomes in the vaccinated subjects in all prospectively defined secondary endpoints, with a significant reduction in the level of colonization by the challenge organism as measured by fecal shedding. Additionally, significant positive impacts of the vaccine on several post hoc secondary endpoints, including signs and symptoms of disease, were observed.
Experimental challenge of human volunteer subjects is a widely used model to evaluate the efficacy of vaccines or other interventions to protect against enteric diseases such as ETEC, Shigella
, and Campylobacter
infection before moving on to large and expensive pivotal phase 3 field trials. In the case of ETEC, the frequency and severity of disease observed in such challenge studies are higher than those seen in naturally acquired illness associated with travel, making them “useful for limited hypothesis testing” (22
) rather than reflecting the expected efficacy of a vaccine in the real world. The most commonly used challenge model for ETEC employs the prototypical CFA/I-positive LT/ST strain H10407 (6
). In a recent review of the use of challenge models to evaluate ETEC pathogenesis and vaccine efficacy (26
), it was clearly shown that the H10407 strain produces significantly more severe symptoms than any other strain evaluated. Recently, the protocol for this model was refined, including an overnight fast before the morning of challenge, to allow reproducible attack rates of more than 70% to be obtained in a total of 35 human subjects following an oral dose of only 2 × 107
). Even at this lower dose, however, the severity of disease induced following challenge is comparable to that seen at the higher dose levels and is greater than with any other challenge strain; diarrheal purges of several liters are common, requiring active intervention with antibiotics and oral and intravenous rehydration support to prevent hypotension or hypovolemia.
Sample sizes in challenge studies are limited by both ethical and logistical considerations. Therefore, challenge trials are not generally powered to see statistically significant differences between treatment groups unless those differences are large. Another feature of challenge studies which may underestimate the efficacy of a vaccine is the use of antibiotics as soon as a subject meets a certain clinical definition, effectively shortening the observation period in which a difference in clinical outcome might be observed. While it might be more informative to avoid treatment to see if vaccination shortens the duration of the episodes, this is difficult to do from an ethical perspective.
The definitions of diarrhea severity in the clinical protocol were in line with similar studies performed under investigational new-drug applications in the United States, based on the U.S. Food and Drug Administration and U.S. National Institutes for Health table of AEs (12
), and were a composite of frequency and volume of loose stools passed in any 24-hour period. During inpatient studies, the frequency component of these definitions is felt to be less meaningful, and so we have additionally evaluated the efficacy of the vaccine against the severity of diarrhea based only on the peak volume passed during any 24-hour period. We suggest this to be a more objective assessment of the clinical impact of the diarrheal episode on individual subjects and more representative of the potential for the vaccine to prevent serious, dehydrating diarrhea in travelers and particularly in children in settings of endemicity.
The vaccine was safe and generally well tolerated, as observed in the previous phase 1 study (17
), with the exception of episodes of vomiting seen in seven vaccinated subjects on the day of vaccination. These episodes were unexpected, given that no such AEs were seen previously. In the phase 1 study, which was the first administration of the multivalent combination to human volunteers, the first dose of vaccine was given on the second day of a 4-day inpatient period as a safety precaution. As a consequence of this, the subjects' diet was carefully controlled; they were given a light breakfast and fasted for approximately 90 min before dosing. In contrast, the subjects in this phase 2b study came into the clinic on the morning of dosing, and many of them had not eaten since the night before (data not shown). It is possible that ingestion of the high dose of vaccine on an empty stomach might have contributed to this reactogenicity. Similar episodes of vomiting postimmunization have been reported for an experimental killed, whole-cell ETEC vaccine (4
). This hypothesis is further supported by the increased virulence of the wild-type challenge with H10407 following overnight fasting (16
). This will need to be addressed during further development of the vaccine by adjusting the buffer formulation and dose level.
The reduction in the primary endpoint of the study was 26.5%, which was not statistically significant. A number of alternative measures of disease severity did, however, reach statistical significance, and others showed strong trends (). shows the incidence of diarrhea by severity based on output alone. The impact of vaccination is spread across the whole range of severity, with each category other than “no diarrhea” having a higher incidence in placebo recipients and with almost three times as many subjects in the ACE527 group being free of diarrheal stools altogether (P = 0.046). In terms of impact on individual subjects, the most significant effect of vaccination was seen on the output of diarrheal stool produced in the first 24 h of an episode, as shown in . This suggests a useful impact of vaccination for travelers, who would typically self-medicate within 24 h of the beginning of a diarrheal episode, which would be expected to prevent worsening of symptoms and accelerate resolution. Therefore, a milder disease course in this first 24 h may mean that the vaccine would reduce the number of travelers whose symptoms interfered with normal activities. This observation is supported by the significantly lower number of vaccinated subjects who considered that their illness would have been sufficiently severe to prevent their normal activities if they were traveling ().
In contrast to some other challenge studies (22
), however, the effect on stool output continued throughout the 5-day observation period after challenge. This effect is readily seen from the graphical representation in . The median number and output of diarrheal stools during this period were reduced by 50% and 65%, respectively ().
When subjects are dichotomized according to the incidence of diarrheal output of greater than 800 g in the 5 days after challenge, there is a 41% reduction in incidence in vaccinees (P
= 0.027). This effect is greater than that on the peak output in 24 h and results from the earlier truncation of output in vaccinees than in placebo recipients. This is clearly seen in ; the median output in the first 24 h of the episode was 310 g in ACE527 vaccinees and increased to 390 g (26% more) by the end of the surveillance period, whereas in placebo recipients the corresponding increase was from 488 g to 1,128 g (131% more). Thus, more placebo recipients continued to produce significant diarrhea after 24 h from the beginning of the episode, despite the fact that many were treated with antibiotics. H10407 has been demonstrated to produce a longer duration of diarrhea than other ETEC strains in challenge studies (26
). It appears from our observations that vaccination with ACE527 truncated this extra duration, perhaps by modulating colonization of the small intestine.
Shedding of H10407 challenge strain.
For the first time, this study showed that vaccination with an oral, whole-cell ETEC vaccine had a significant impact on the quantitative level of colonization of subjects as measured by fecal shedding. Geometric mean concentrations of H10407 shed at 2 days after challenge were reduced by 20-fold; this level of reduction in vaccinees is comparable to that seen in other studies.
In a study in which subjects were fed CFA-specific antibodies or a placebo in milk before challenge with H10407 (14
), the mean level of the challenge organism was 6.2 × 107
CFU/g in milk globulin-treated subjects versus 4.5 × 108
in placebo recipients, a 7.3-fold reduction. Treated subjects experienced 90.5% (95% CI, 33.9% to 98.6%) protection against diarrhea. In a study aimed at refining the H10407 challenge model, subjects fed 2 × 107
CFU shed geometric mean maximum concentrations of 8 × 107
CFU/g, whereas subjects rechallenged with the same dose shed only 3 × 105
), a 270-fold reduction, and were protected against diarrhea.
In a previous ETEC vaccination challenge study using a spontaneous toxin-deleted ETEC strain (E1392-75/2A) as a vaccine and a different ETEC challenge strain (E24377A) (19
), 75% protection against diarrhea was seen, and a reduction in colonization by the challenge strain was observed in vaccinees as measured in small-bowl aspirates. In that study, there was no difference seen in the level of stool shedding, although it is not clear from the literature at what times stool samples were assayed for shedding after challenge. The authors hypothesized that vaccination with the live vaccine strain had induced protective immune responses in the small intestines of immunized subjects and that this interfered with colonization of this niche and the subsequent induction of diarrheal disease. In the present study, a significant reduction was seen in the level of shedding of H10407 in stool on day 2 after challenge in vaccinees, whereas by day 4 their level of shedding, although still lower, approximated that in placebo recipients. We hypothesize that this corresponds to an inhibition of small intestinal colonization early after dosing in vaccinees, followed by a later colonization of the large intestine equally in both groups, as previously described by Levine and colleagues (19
). As colonization of the large intestine by ETEC does not cause diarrhea, a vaccine does not need to inhibit this to exert a protective effect against disease.
Given the highly significant reduction in early shedding of H10407 observed in the vaccine group, it is important to investigate whether this has any functional impact on the incidence or severity of disease. All subjects from whom we were able to collect quantitative shedding data at 2 days after challenge (23 of 29 vaccinees and 24 of 27 placebo recipients) were grouped according to whether their shedding was above or below the median level (5 × 106 CFU/g). The time to reach the primary endpoint of moderate to severe diarrhea (composite definition based on volume or number of loose stools in 24 h) was investigated for these groups using a Kaplan-Meier survival analysis, as shown in . Subjects who did not meet the endpoint during the 120 h following challenge were censored. The hazard ratio for meeting the primary endpoint in the high-shedding group was 2.98 (95% CI, 1.51 to 6.74%), and the survival curves were statistically different (P = 0.002 by the log rank test). Thus, it has been clearly shown that vaccination with ACE527 significantly reduces the shedding of H10407 after challenge and that in subjects with lower shedding there is a highly significant reduction in the odds of meeting the primary endpoint of the study.
Fig 6 Kaplan-Meier survival analysis of the time to reach the primary endpoint (moderate or severe diarrhea) according to the quantitative level of shedding of H10407 in stool at 2 days after challenge. All subjects for whom quantitative shedding data were (more ...)
The subgroup of subjects who mounted positive mucosal responses (IgA ALS) to both CFA/I and LT-B (n = 11, all of whom were ACE527 recipients) showed a significant reduction in the level of shedding of H10407 in stool at day 2 after challenge compare to subjects who responded to neither antigen (n = 22) (P = 0.009) (data not shown). This may indicate the involvement of these specific responses in the reduction of colonization by H10407, or else they may simply be markers of having received the ACE527 vaccine, as there was a significant reduction in shedding in the vaccine group as a whole.
Immune response to H10407 challenge strain.
Vaccination with ACE527 reduced the rate of IgG seroconversion to the O78 LPS antigen expressed by the H10407 challenge strain, most obviously in the early response at day 7 after challenge. This correlates with the impact of vaccination on the colonization of subjects by the challenge strain and provides additional evidence that the interaction of H10407 with the mucosal surface and immune system was impaired in those subjects who were vaccinated with ACE527.
Comparison with previous challenge studies.
Previously, as part of the development of the live attenuated vaccine approach, a similarly attenuated CS1- and CS3-expressing strain of ETEC (PTL003, a derivative of E1392-75/2A lacking any LT toxin antigen) was evaluated in phase 1 and phase 2 studies (21
), which failed to show efficacy against challenge with virulent strain E24377A (LT, ST, CS1, CS3) in the latter. That challenge trial was performed with a different immunization schedule (2 doses 10 days apart), a lower vaccine dose (2 × 109
CFU), and a higher challenge dose (3 × 109
CFU), and the vaccine contained no LT antigen. The results of this trial may therefore not be relevant to the potential efficacy of ACE527, which contains three different ETEC strains and induces strong immune responses against LT. In the study reported here, we addressed these shortcomings by increasing the vaccine dose, leaving a longer interval between doses, including LT, and using a low-dose refined challenge protocol. As a result, we demonstrated a clear reduction in many of the parameters of ETEC diarrhea severity as well as a trend toward a reduction in the incidence of moderate to severe diarrhea, the primary endpoint of the study, and a statistically significant reduction in the shedding of the challenge organism. It should be noted that this is the first time that such protective efficacy has been seen in an ETEC challenge study with a multistrain vaccine, where only one of three vaccine strains matches the CFA type of the challenge organism.
This contrasts with another recent challenge study, performed using challenge strain E24377A, with a vaccine comprising LT delivered in a skin patch (22
). Although there were reductions in the number and volume of stools following challenge in subjects vaccinated with the patch, this effect was concentrated in the 24 h following challenge, after which the groups became more similar, and there was no impact on the overall incidence of moderate to severe diarrhea. As expected for a vaccine lacking colonization factors, there was no impact on fecal shedding of the E24377A challenge strain. Despite this modest impact in an inpatient challenge setting, however, the LT patch vaccine went on to show impressive efficacy against traveler's diarrhea in a phase 2 trial (13
) and against diarrhea caused by LT ETEC strains in an expanded phase 3 trial (18
). This suggests that ACE527 may show similar or higher efficacy in a field setting, where subjects are exposed to significantly lower levels of ETEC, are usually not fasted, and do not protect the ETEC organism by buffering the stomach pH. In such settings subjects are exposed to a variety of ETEC strains expressing multiple CFA and toxin types, most of which may be of lower virulence than H10407.
This phase 2b study was designed to provide early evidence for the efficacy of ACE527 using a preliminary formulation and a high dose considered to be appropriate for controlled clinical trials. While the study population may be pertinent to the future development of the vaccine for use in travelers from countries where ETEC is not endemic to less-developed regions, it is not expected to be particularly relevant to the evaluation of either the safety or efficacy in pediatric populations in developing countries. These parameters can be studied only in the appropriate target populations. ACE527 induced clinically significant attenuation of diarrheal illness and reduced ETEC intestinal colonization in a stringent ETEC H10407 human challenge model. The reduction in the shedding of H10407 following vaccination with ACE527 and the strong induction of anti-LT immune responses support the dual mechanism of action that the vaccine was designed to exhibit. This dual mechanism suggests that the vaccine may prove to be effective against the majority of wild-type ETEC strains encountered in cases of natural exposure, although this remains to be proven in future field trials. The vaccine was safely administered orally at a total dose of 1011 CFU, though further refinements of dosage and formulation are warranted, in conjunction with expanded trials to demonstrate safety, tolerability, and efficacy in at-risk populations. In preparation for future field testing, a follow-on study in healthy adults was recently initiated to evaluate the safety, tolerability, and immunogenicity of a new lyophilized formulation of ACE527, administered at a lower dose, with or without a mucosal adjuvant and according to a dosing schedule more appropriate for pediatric immunization schedules.