This study demonstrates that a single oral dose containing 109
CFU of freshly cultured V. cholerae
638 is well tolerated, immunogenic and able to afford short-term protection (at 1 month) against cholera in a volunteer model that uses strain V. cholerae
3008 as the challenge agent. This model has been previously used at the Center for Vaccine Development (Maryland) to assess protection conferred by El Tor Ogawa strain CVD111 (25
). Clinical and bacteriological responses to V. cholerae
3008 in challengees of the placebo group in the present study were similar to those seen by Tacket et al. (25
), in unimmunized North American volunteers. The attack rate of diarrhea in North Americans was 7 of 8, while in Cuba it was 7 of 9; the mean diarrheal stool volume in North Americans was 2,534 ml (984 to 7,703), and in Cuba it was 2,231 ml (330 to 5,731). The mean number of diarrheal stools was 14 in both studies, ranging from 6 to 46 in the United States and from 3 to 34 in Cuba. The mean peak stool excretion levels were 9.3 × 106
CFU/g in Maryland and 2.3 × 106
CFU/g in Cuba. In both studies, all nonvaccinated volunteers exposed to 3008 seroconverted with vibriocidal antibodies with mean peak reciprocal titers of 2,177 (United States) and 2,240 (Cuba). We interpret from this analysis that Cuban volunteers are similarly susceptible to infection and cholera produced by wild-type strain 3008.
In this model of challenge with strain 3008, V. cholerae
638 provided significant protection against fecal shedding of the challenge agent and complete protection against cholera (any diarrhea) (Table ). Protection against severe and moderate cholera could not be rated since only two subjects in the placebo group met the definition of severe cases. As an additional criterion for protection, challenge did not boost the titers of vibriocidal antibodies in the vaccinees but induced 100% seroconversion in challengees of the placebo group (Tables and ). These results agree with previous data in which the titers of vibriocidal antibodies of volunteers orally inoculated with the live V. cholerae
O1 vaccine strain CVD 103-HgR were not significantly increased after second administration of the same strain one year later (27
) or after challenge with fully virulent strains of V. cholerae
within 4 months (17
In contrast, the counts of IgA-ASC in all volunteers from the vaccine group had slight but detectable postchallenge increases (Table ). Since the challenge agent was not fecally shed by most of the vaccinees (75%), this likely represents a limited process of antigen sampling at mucosal priming sites during gut passage of the challenge agent that lead to low responses. These results contrast with previous findings (27
) in which volunteers primed with CVD103-HgR and boosted 418 days later with the same strain did not respond with IgA-ASC following reexposure. One important difference here is that reexposure was done with the wild-type challenge agent V. cholerae
3008 and not with an attenuated strain. Because of the lack of extensive work addressing this topic, it merits further analysis in future studies. In correspondence with the increase in IgA-ASC in blood, the level of circulating anti-LPS IgA also increased by 2-fold or greater in 7 of 12 challengees of the vaccine group (Table ).
The protection afforded by vaccination with 638 was accompanied by a 96% seroconversion rate for vibriocidal antibodies, the best indirect marker of protection. Equally, high percentages of specific IgA-ASC (100%) and salivary IgA (75%) producing individuals were detected among the vaccinees. However, the numbers of specific IgA-ASC were higher after feeding the virulent strain than after immunization with attenuated strain 638 (mean numbers of ASC per 106 PBMC, 1,063 and 248, respectively). This may result from the positive effect exerted by cholera toxin on the immune response to V. cholerae 3008 or from the level of attenuation attained during construction of 638.
The immune response to vaccination with 638 resulted from colonizing activity of the attenuated agent as revealed by detection of fecal shedding of vibrios. Shedding did not begin the day after vaccination in all volunteers, which led to a median incubation period to excretion of 3 days. This result contrasted with the one obtained for the progenitor strain V. cholerae
81 and the toxigenic challenge strain 3008 for which the incubation period to shedding was 1 day in all volunteers, and most subjects kept sustained excretion for the entire 5-days follow-up period. It is not completely clear whether the longer incubation period to excretion seen with 638 in humans is due to the presence of an inactivated hapA
gene and the consequently lower detachase activity (3
) or due to an occult spontaneous mutation that may have occurred during construction of strain 638. The wild-type hapA
gene has been restored to strain 638 in order to test the effect of Hap on safety and vibrio excretion in volunteers (unpublished data).
Strain 638 induced systemic responses of vibriocidal antibodies in 96% of the volunteers without inducing relevant adverse events. These results are in agreement with previous volunteer studies that addressed the safety and immunogenicity of the attenuated strain (3
). Here, the most important symptom elicited was grade 3 diarrhea in 4 (16%) of 24 vaccinees in comparison with 2 (10%) of 21 placebo recipients, which resulted in nonsignificant differences in the numbers of episodes or their intensity rates. In contrast with strain 638, its progenitor strain, V. cholerae
81 induced diarrhea in 46% of the subjects. Thus, the safety of strain 638 is not a common feature in all C7258 derivatives, but an inherent attribute of strain 638, presumably due to inactivation of hapA
with the marker gene celA
In this work, we exploited the high rates of fecal shedding and reactions induced by strain 81 to make an exploratory challenge with this attenuated agent to subjects vaccinated with strain 638. Our findings indicated that vaccinees with strain 638 were protected from infection and symptoms elicited by 81 and allowed us going into the wild-type challenge studies. This small study is of interest, since in the future one could consider using an attenuated but still colonizing and reactogenic mutant as a more convenient challenge strain to measure protection to infection without the need to induce cholera in subjects of the placebo group. This would be possible because it is widely accepted, although not conclusively established, that protective immunity to cholera is mainly mediated by secretory IgA antibodies directed against bacterial antigens more than an antitoxic response (14
The results of the present study indicate that strain 638 is safe, immunogenic, and protective in healthy volunteers. Future studies with lots prepared under good manufacturing practices for clinical trials should address the protective capacity conferred by 638 3 months and later after vaccination of volunteers. Subsequent field studies should evaluate whether the final vaccine preparation is protective in areas of endemicity.