Cholera affects all age groups. In zones of endemicity, young children are vulnerable (10
), and by adolescence most have serological evidence of previous exposure to V. cholerae
). During outbreaks of cholera in zones where cholera is not endemic, children and adults are equally affected by cholera (21
). As such, children bear a very large burden of cholera globally (10
). Despite this, current oral cholera vaccines are associated with lower protective efficacy and a shorter duration of protection in younger children than observed following vaccination of adults (7
). In addition, vaccination against cholera is also associated with lower levels of protection and a shorter duration of protection than wild-type infection with V. cholerae
O1 provides (19
), despite the fact that protection by wild-type infection against subsequent cholera appears to be similar in younger children and older persons (4
). The immunologic correlates of these differences are poorly understood, especially in children. Here, we show that immune responses to an oral killed cholera vaccine (WC-rBS) are generally comparable in younger and older children in an area where cholera is endemic; however, these responses are much lower than those detected following wild-type cholera infection, most notably in anti-LPS responses in plasma, the vibriocidal response, and memory B cell responses to LPS.
The vibriocidal antibody is the most-studied immunologic marker of cholera infection and is an indirect/surrogate marker of protection. While vibriocidal titers correlate with protection against cholera, there is no threshold at which protection is complete (34
). The vibriocidal response can be largely removed by adsorption of plasma to LPS, strongly suggesting that antibodies reacting with LPS are a critical component of the vibriocidal response (26
). Few direct comparisons have been made of the differences in vibriocidal responses to OCV between younger and older children. In immunogenicity studies of WC-rBS in Peruvian children, younger and older children had comparable seroconversion rates 14 days after the second dose of vaccine (39
). In our study, we similarly found no differences in vibriocidal responses between younger and older vaccinated children although a subanalysis disclosed that the youngest children (defined as ≤4 years of age) were less able to mount a vibriocidal response than older children. However, we did find that age-matched patients with V. cholerae
O1 Ogawa infection had significantly higher responses (both in magnitude and responder frequency) than vaccinees at all days evaluated.
We have previously shown that LPS and CtxB plasma IgA antibody responses correlate with protection against cholera in household contacts of cholera patients (14
). In our current study, we now show that two doses of WC-rBS vaccination induce prominent IgA and IgG anti-CtxB responses in younger and older pediatric vaccine recipients and that these responses are generally comparable to those induced in children with wild-type cholera infection, differing only in day 30 IgG and day 7 IgA responses in the younger children. These similarities may in part reflect that each dose of WC-rBS is supplemented with 1 mg of recombinant CtxB. It should be noted that an alternative OCV, bivWC (Shanchol), does not contain supplemental CtxB, and the anti-CtxB responses induced by WC-rBS (Dukoral) and bivWC (Shanchol) may well be different. Despite our ability to detect prominent anti-CtxB responses following vaccination of children with WC-rBS and after wild-type disease, we found a marked difference between anti-LPS plasma responses in vaccinees versus children recovering from wild-type cholera disease in both age groups. Specifically, although vaccination induced low-level plasma IgG, IgA, and IgM responses against LPS that were similar in younger and older children, these responses were much lower than those observed following wild-type disease in age-matched controls.
Immunity against cholera following wild-type disease is believed to last at least 3 to 10 years (4
). Since acute-phase immune responses including vibriocidal and plasma antibody responses fall to baseline within 6 to 12 months of infection (13
), longer-term protection against cholera is probably afforded by the ability to mount anamnestic responses facilitated by long-term memory responses (17
). We have previously found that anti-LPS and anti-CtxB memory B cell responses are induced following wild-type cholera infection and that these responses persist even after the vibriocidal and plasma antibody responses have returned to baseline (13
). In adults, we have also demonstrated that oral cholera vaccination with WC-rBS induces anti-CtxB memory B cell responses but not anti-LPS memory responses (2
In our current analyses, we have now extended this work. When results were analyzed by age cohort, we were unable to detect anti-LPS or anti-CtxB memory B cell responses in both younger and older vaccine recipients although when we analyzed in aggregate, we did detect IgG memory B cell responses against CtxB in children receiving WC-rBS. In comparison, we were able to detect significant MBC responses against CtxB and LPS in children recovering from cholera when responses were analyzed both by age cohort and in aggregate. The ability of both naturally infected and vaccinated children to generate anti-CtxB memory responses may in part reflect that the cholera vaccine used in this study contains 1 mg of recombinant CtxB per dose, as well as the potential boosting of preexisting immune responses to the immunologically cross-reactive heat labile toxin (LT) of enterotoxigenic E. coli
), an infection that is endemic in resource-limited areas of the world including Bangladesh (30
). However, the significance of these CtxB responses in protecting against cholera is uncertain since there is no heterologous protection against cholera between V. cholerae
O1 and O139, despite the fact that that these serogroups express identical enterotoxins (29
), and since protective efficacy of oral cholera vaccines containing and not containing supplemental CtxB are comparable (7
No difference has been noted in the degree of protection against subsequent disease afforded by previous cholera infection in younger and older children in areas of endemicity (4
); however, following vaccination, young children have a significantly shorter duration and lower level of protection than older persons (7
). In our current study, we found that the plasma antibody against LPS, the memory B cell response against LPS, and vibriocidal responses (largely targeting LPS) are significantly lower in child vaccinees than in children recovering from wild-type cholera infection. This is particularly significant when we consider that responses against LPS may be a prime mediator of protection against cholera (11
LPS is a T cell-independent antigen (16
), and young children are less able to mount T cell-independent responses than older children and adults (18
). Our inability to detect differences in acute and MBC responses against LPS in comparing cohorts of younger and older children following vaccination in this analysis may in part reflect the relatively small size of our vaccine cohorts and the fact that the mean age of our cohort of ≤5 years of age was 4.7 years. As such, many of these young children in this area of high endemicity for cholera may already have been exposed to V. cholerae
), mitigating our ability to detect differences in younger versus older vaccine recipients. This possibility is further supported by the fact that we were able to detect a significant increase in both plasma IgG and IgA to both CtxB and LPS within 3 days of administration of oral cholera vaccine even in the cohort of children ≤5 years old in this study, suggesting prior exposure and a primed response. Similarly, the observation in our subanalysis that the youngest vaccinees in this study were less able to mount a vibriocidal response than older vaccinees supports this hypothesis. Whether we would have found similar results if we had conducted the study in an area where cholera is not endemic or if we had focused our enrollment and analysis on children less than 3 years of age is uncertain. We also cannot exclude the possibility that vaccination induces low-level memory responses against LPS in children but that these responses are below the level of detection of our assay. Finally, our inability to specifically detect differences in anti-LPS memory B cell responses between older and younger vaccine recipients may also be due to the fact that oral cholera vaccines are particularly poor inducers of LPS-specific memory B cell responses, even in immunologically primed adults (2
), and this reality complicates our ability to detect differences between younger and older children for this immunologic marker.
A potential shortcoming of our study is that our group and others have demonstrated the effect of zinc supplementation (1
), small bowel bacterial overgrowth (20
), and antiparasitic drug treatment (8
) on immune responses to OCV in young children. We did not assess differences between age groups of such modifiers of immune responses in this study, and these factors deserve further investigation. Furthermore, difficulties in recruiting child participants, especially those less than 4 years of age, limited our sample size.
In conclusion, in this study we demonstrate that both younger and older children vaccinated with a WC-rBS oral killed cholera vaccine formulation mount comparable immune responses in this area of high endemicity for cholera where prior exposure may have occurred but that these responses, especially vibriocidal and LPS responses, including memory B cell responses, are significantly lower than the responses detected in age-matched children recovering from natural cholera infection requiring hospitalization. These observations are significant and may in part explain differences in protective efficacy and duration of protection observed between wild-type infection and vaccination with currently available oral cholera vaccines.