The present study is the first head-to-head study comparing immune responses to Vi and Ty21a vaccines, the two typhoid vaccines currently available. We found similar numbers of typhoid-specific plasmablasts in the circulation of the vaccinees in both groups, yet the homing profiles were found to be different. As an unforeseen finding, the antigen-specificity of these responses proved less different than had been thought.
Until now, no studies have explored circulating Vi-specific plasmablasts in volunteers receiving typhoid vaccines. A clear Vi-specific response was mounted in the Vi group, while none in the Ty21a group responded to this antigen, in alignment with the lack of Vi antigen in the Ty21a strain. In the Ty21a group, the highest response was mounted to O-9,12 antigen. No response to O-9,12 was initially anticipated in the Vi group, yet O-antigen-specific plasmablasts were found in almost all vaccinees. Even if significantly lower than in the Ty21a group, the response was clear.
The response to the typhoidal O-antigen in the Vi group calls for an explanation. A logical reasoning derives from the fact that the Vi polysaccharide was initially isolated from the Salmonella
Typhi Ty2 strain. Since it is not an easy task to remove the contaminating lipopolysaccharide (LPS), traces of typhoidal LPS are likely to be left in the final preparation. This amount appears small enough not to cause adverse effects typical to parenteral whole-cell typhoid vaccines, yet high enough for the immune system to recognize it. In fact, in early studies, serum antibodies to LPS had been reported in Vi vaccinees, the levels correlating positively to the LPS content of the preparation: 5% of LPS caused seroconversion in 83% of vaccinees, and 0.2% of LPS in only 26% 
In the present study, no immune response to H-d antigen was observed. In our previous study, a plasmablast response against H-d antigen was found after parenteral whole-cell Ty21a, but not after oral Ty21a vaccination 
, suggesting immunization route-dependent differences in processing the antigen. In the present study, the lack of response to H-d found in the Vi group suggests that either the antigen has been destroyed in the vaccine purification process, or the amount that is left of it remains too low to elicit a measurable response.
Previous studies with Ty21a vaccines have shown that the numbers of plasmablasts 7 days after vaccination correlate with levels of effectiveness observed in field trials using the same immunization regimen 
. As the Vi and Ty21a vaccines have in various field trials been reported to confer a similar degree of protection, we were curious to know whether they elicit similar levels of typhoid-specific plasmablasts in the circulation. The total typhoid-specific response was estimated with two separate approaches: by using whole-cell S.
Typhi as antigen in the assay, and by calculating the total of plasmablast responses to all typhoidal antigens measured, i.e. to Vi, O-9,12, and H-d antigens. In the Ty21a group these two approaches yielded identical results, whereas in the Vi group the former approach appeared less sensitive than the latter. We suggest that using whole-cell S.
Typhi as an antigen may not allow a sufficiently high concentration of the Vi antigen in the assay or, alternatively, the Vi polysaccharide may have been partly destroyed by formalin, when preparing the strain for the assay. Therefore, the response to Vi antigen should be assessed separately, as was done in the latter approach. The total of the response to the different typhoidal antigens (Vi+O-9,12+ H-d) was found to be similar in the two vaccine groups, consistent with the similar efficacy of these vaccines in field trials 
. Interestingly, the magnitude of the response in a natural typhoid infection (for three patients mean specific ASC/106
PBMC against S
. Typhi 343, O-9,12 216, H-d 3 and Yersinia enterocolitica
3) in our previous studies 
appears similar to that reported for the two vaccine groups in the present study.
Immunity to typhoid fever is considered to consist of several factors, including both cell-mediated and humoral immune mechanisms 
. The conception that the immune mechanisms of these two vaccines are totally different would imply that typhoid immunity can be attained in at least two entirely different ways: through antibodies against Vi antigen (as with Vi vaccine) or by several other mechanisms, e.g. by eliciting cell-mediated immune responses, by specific intestinal secretory IgA, and LPS-specific serum antibodies (Ty21a vaccine). However, an O-antigen-specific plasmablast response also conferred by the Vi vaccine suggests that the current conception of the protective mechanisms of this vaccine may need to be revisited. O-antigens are considered to contribute to the defence conferred by whole-cell parenteral vaccines: protective efficacy has been reported by vaccines prepared both from Vi-positive (K strain) and -negative (L strain) S.
Typhi strains 
. Interestingly, Citrobacter
5396/38 and S.
Typhi have identical Vi antigens. In early studies, Vi polysaccharide isolated from the Citrobacter
strain was found to be less effective than the one isolated from S.
. The authors wondered whether the isolation of Vi antigen had been less successful from the Citrobacter
. Typhi Ty2 strain. One might also argue that only the latter preparation contained typhoidal LPS. Similarly, it thus appears possible that the protection conferred by the current Vi vaccines, while mainly depending on Vi-specific mechanisms, could also include mechanisms specific to the O-antigen part.
The homing profile of the plasmablasts reflects the expected localization of the immune effector cells in the body. The plasmablasts in the Vi group exhibited a systemic homing profile, with a high frequency of L-selectin- and a lower frequency of α4
- and CLA-expressing cells. In the Ty21a group, by contrast, the homing profile was intestinal 
, with all cells expressing α4
and a lower proportion L-selectin and CLA. These results are consistent with previous studies on oral 
and parenteral 
antigen administration showing that the expression of HR depends on the site of antigen encounter 
. Both intestinal and systemic immune defence is needed in the protection against typhoid fever. The intestinal targeting appears particularly beneficial from the perspective that the pathogen uses this site as a portal of entry, a fact reflected as a gut-directed HR profile in patients with a natural typhoid infection. These data complement the view that in contrast to Vi, vaccination with Ty21a closely imitates the natural typhoid infection.
Serum antibodies are the classical approach to studying humoral immune responses. While serum antibodies provide information on the systemic immune response, the plasmablast assays (ELISPOT and ALS) describe the total response elicited in the body. Consistent with previous studies 
, the ELISPOT assay assessing the response at single-cell level proved to be the most sensitive approach to the evaluation of the response.
Consistent with the level of protection reported in field trials, the magnitude of the typhoid-specific plasmablast responses to Vi and Ty21a vaccines are similar. However, there is a difference in the expected localization of the response, and the antigenic specificity. Only the response to Ty21a vaccination was found to imitate closely that in a natural infection. Surprisingly, the antigen specificity assays also revealed a significant O-antigen-specific response in the Vi group, presumably caused by trace amounts of lipopolysaccharide remaining in the vaccine preparation after purification. It remains possible that part of the immunity conferred by the Vi vaccine may, in fact, be contributed by a response to typhoidal O-antigens.