HUMAN IMMUNOLOGY doi: 10.1111/sji.12151 ..................................................................................................................................................................
Cross-Reactive Immune Response Induced by the Vi Capsular Polysaccharide Typhoid Vaccine Against Salmonella Paratyphi Strains S. H. Pakkanen*†, J. M. Kantele‡ & A. Kantele*†§
Abstract *Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; †Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland; ‡Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland; and §Department of Medicine, Institute of Clinical Medicine, University of Helsinki, Helsinki, Finland
Received 16 November 2013; Accepted in revised form 22 December 2013 Correspondence to: Adjunct Professor Anu Kantele, Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, POB 348, FIN-00029 HUS Helsinki, Finland. E-mail: [email protected]
There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross-reactive immune response against S. Paratyphi A and B, and some reports suggest cross-protective efficacy against the disease. These findings are ascribed to the O-12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O-9,12. Twenty-five volunteers immunized with the parenteral Vi vaccine (Typherixâ) were explored for plasmablasts cross-reactive with paratyphoid strains; the responses were compared to those in 25 age- and gender-matched volunteers immunized with Ty21a (Vivotifâ). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi- and Ty21a-vaccinated volunteers had circulating plasmablasts producing antibodies cross-reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross-reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi-negative, the result can be explained by trace amounts of bacterial cell wall O-12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined.
Introduction There is an urgent need for vaccines against paratyphoid fever, as both the incidence of the disease [1–5] and antimicrobial resistance are increasing [1, 3, 6–9] and there are no vaccines available. Paratyphoid fever is caused by Salmonella enterica subsp. enterica serovar Paratyphi A and B (S. Paratyphi A and B) and, rarely, Salmonella enterica subsp. enterica serovar Paratyphi C (S. Paratyphi C). The global number of paratyphoid fever cases has been estimated at 5.4 million cases per year [10]. Recently, it has been proposed that the oral live attenuated Ty21a typhoid vaccine (Vivotifâ) could be used as a surrogate vaccine against paratyphoid fever until a vaccine specifically targeted against this disease becomes available [11]. This line of thinking is based on reports of Ty21a vaccine conferring cross-protective efficacy against paratyphoid fever [12, 13] and, consistent with them, on immunological studies
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attesting that Ty21a-vaccinated volunteers develop substantial cross-reactive cell- [14–16] and antibody-mediated [11, 17] immune responses against S. Paratyphi A and B. Cross-protection [12] and cross-reactive immune response [11, 14, 17] elicited by the Ty21a vaccine have been explained by the common O-12 antigen found both on S. Typhi, Ty21a vaccine strain, and on S. Paratyphi A and B. Accordingly, the Ty21a has been shown to elicit a significant plasmablast response to all these strains, but not against S. Paratyphi C and S. Egusi, which carry unrelated O antigen types (O-6,7 and O-41, respectively) [11]. The Ty21a and the Vi capsular polysaccharide vaccines have different antigenic compositions [7, 18]. The Ty21a is a whole-cell vaccine sharing the O antigens 9 and 12 with S. Typhi, but lacking the Vi polysaccharide, whereas the Vi vaccine is a purified polysaccharide preparation that has been considered to share only the Vi antigen with S. Typhi. While the Ty21a vaccine elicits a significant cross-reactive
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immune response solely against S. Paratyphi A and B [11], the Vi vaccine is expected to induce a response only to S. Paratyphi C which, in contrast to S. Paratyphi A and B, carries the Vi antigen. However, as a preparation initially isolated from the S. Typhi Ty2 strain, the Vi vaccine contains traces of contaminating typhoid lipopolysaccharide with O-9,12 antigens. In our recent study, we demonstrated that this amount suffices to elicit an immune response [19]. This was found by exploring plasmablasts appearing in the circulation after vaccination. They represent maturing B cells trafficking from the initial site of antigen encounter via blood to sites where they settle down as plasma cells [20]. Assessing circulating plasmablasts has proved to be an especially sensitive approach to evaluating the humoral response to vaccinations, either by oral [19, 21–28] or parenteral route [19, 22, 24, 27, 28]. The recent observation of O antigen-specific humoral immune response induced by the Vi vaccine raises a question of whether this vaccine could elicit a cross-reactive immune response against S. Paratyphi A and B, consistently with the Ty21a vaccine.
Material and methods Ethics statement. The study was conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved both by the ethics committee of the Helsinki University Central Hospital and the Finnish Medicines Agency and entered in the registry of Current Controlled Trials Ltd. c/o BioMed Central (International Standard Randomized Controlled Trial Number ISRCTN68125331). Written informed consent was obtained from all study subjects. Study design. Cross-reactive immune response to S. Paratyphi strains was explored in volunteers vaccinated with Vi polysaccharide (Typherixâ; GlaxoSmithKline Biologicals s.a., Rixensart, Belgium) and compared to that in age- and gender-matched volunteers vaccinated with the live oral typhoid S. Typhi Ty21a (Vivotifâ; Crucell, Leiden, Netherlands) vaccine. Circulating plasmablasts specific to S. Paratyphi A and B (O-12 antigen in common with S. Typhi) and S. Paratyphi C (Vi antigen in common with S. Typhi) were studied by enzyme-linked immunospot assay (ELISPOT) before and 7 days after administering the Vi vaccine or the first dose of Ty21a to identify cross-reactive antibody-secreting cells (ASC). For comparison, data on immune responses to the various typhoid antigens and Yersinia enterocolitica (negative control) in both of these vaccination groups were retrieved from our recent report [19], and responses to paratyphoid strains were compared to these. Results of the paratyphoid-specific responses in 17/25 volunteers in the Ty21a group have been incorporated in our recently published data, where no age- and gender-matched controls were included [11]. The present study only included age- and gender-matched
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volunteers who had initially been randomized to receive either the Ty21a or the parenteral Vi vaccine. The study was conducted at the Helsinki University Central Hospital and at the Haartman Institute, University of Helsinki between December 2009 and October 2010. Volunteers, vaccinations and samples. Fifty healthy volunteers were randomized into two age- and gender-matched groups of 25 volunteers (both groups consisted of 17 females and 8 males aged between 22 and 62; average 32 years) with no history of typhoid vaccination or typhoid or paratyphoid fever, the numbers of volunteers were evaluated in prestudy power calculations based on responses in pilot experiments. A parenteral Vi polysaccharide vaccine (Typherixâ; GlaxoSmithKline Biologicals s.a., lots ATYPB084BC and ATYPB096AF) was given to one group, and a live oral vaccine containing ≥2 9 109 live Salmonella Typhi Ty21a bacteria (Vivotifâ, Crucell, lot 3001777) to the other. The amount of contaminating endotoxin in the Typherix vaccine preparation was 13.30 EU per dose for lot ATYPB096AF and 27.00 EU per dose for lot ATYPB084BC (data provided by the manufacturer). The parenteral vaccine was administered with a 25-mmlength needle as one 0.5 ml dose in the left deltoid muscle of the volunteers on day 0. The oral vaccine was administered as one enteric-coated capsule on days 0, 2 and 4, following the manufacturer’s recommendation. The vaccine type given to the first volunteer of each age was determined by raffle, and the second, age- and gendermatched subject, was given the other vaccine type. Notably, the results of the assays on the coded ELISPOT plates were read by a blinded person with no knowledge about the types of vaccine given in the various cases or which plates represented age- and gender-matched pairs. Both vaccines proved well tolerated, as reported in our previous study where all adverse effects are described [19]. There were no dropouts in the study. A sample of peripheral venous blood was drawn on days 0 and 7 after administering the Vi vaccine or the first dose of Ty21a. Previous studies of mucosal [21, 28, 29] and parenteral [28] antigen administration show that the numbers of ASC peak on day 7 after antigen encounter, and the cells disappear by day 14–16. Accordingly, day 7 was chosen as the time point for catching circulating ASC. Antigen. Strains of S. Paratyphi A, B and C were used for coating in the ELISPOT assay. The results were compared to data retrieved from our previous study exploring in the same volunteers the response against S. Typhi and Yersinia enterocolitica (negative control). The total response to S. Typhi had been calculated by adding together the response to typhoidal O-9,12 antigen (represented by Salmonella SL2404 strain), Vi antigen and H-d (represented by S. Egusi) [19]. The S. Paratyphi A (RHS6716) and B (RHS6744) strains were obtained from the collection of the National Institute for Health and Welfare (THL), Helsinki, Finland,
224 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. and the S. Paratyphi C strain from the ACTT (ATCC13428; American Type Culture Collection, Manassas, VA, USA). To confirm the expressions of the O and Vi antigens, each bacterial strain was analysed by the Finnish national reference laboratorium of the THL according to their routines. The preparation of the antigen has been explained in detail previously [11, 21]. ELISPOT assay of specific ASC. PBMC were separated using Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) centrifugation of fresh heparinized venous blood [21]. These cells were explored with ELISPOT for ASC specific to the various paratyphoid strains, as described previously [15]. In brief, 96-well microtitre plate wells (Maxisorp, Nunc, Roskilde, Denmark) were coated with a whole-cell preparation of formalin-killed bacteria. The PBMC were incubated in the wells for 2 h, and antibodies secreted during this time were detected with alkaline phosphataseconjugated goat anti-human IgA (Sigma-Aldrich, St. Louis, MO, USA), IgG (Sigma-Aldrich) and IgM (SouthernBiotech, Birmingham, England). The substrate (bromo-4chloro-3-indolyl phosphate p-toluidine salt; Sigma-Aldrich) was added in melted agarose. The spots were enumerated with AID Elispot reader, and each spot was interpreted as a print of one ASC. The specificity, linearity, stability and intermediate precision of the ELISPOT assay have all been validated (data not shown). A responder was defined as a person with at least 3 IgA+IgG + IgM-ASC/106 PBMC and marked as LOD (limit of detection) of the response in the figures. This limit has been determined over the assay validation process. Statistics. Statistical analyses were carried out with JMP software version 9.0.0 (SAS Institute Inc, Cary, NC, USA). After logarithmic transformation of the data, the distributions of the numbers of ASC were tested with Shapiro– Wilk’s test. All proved normal. Student’s t-test was used for comparisons, applying the Bonferroni correction to various antigens. Differences were considered significant when P < 0.05. The results are given as the means and 95% confidence intervals (CI) for the number of ASC.
Results Antibody-secreting cells response in the Vi group
Before vaccination, no S. Paratyphi A/B or C-specific ASC were found in the circulation of 24/25 vaccinees in the Vi group (Fig. 1). One volunteer, who had 1 week earlier had an upper respiratory tract infection, had 98 ASC/106 PBMC to S. Paratyphi A, 95 to B and 60 to Paratyphi C (Fig. 1). We have previously shown that infections can be associated with a certain degree of polyclonal immune response [30, 31]. On day 7, circulating ASC reactive with S. Paratyphi A, B and C were found in 15/25, 18/25 and 16/25 volunteers
(Fig. 1). The magnitude (mean with 95% CI) and statistical comparisons between the responses to the various strains are shown in Table 1. IgA-, IgG- and IgM-ASC were equally presented in the cross-reactive responses in the Vi group (Fig. 2). Antibody-secreting cells response in the Ty21a group
Before vaccination, no S. Paratyphi A or B -specific ASC were found in the circulation of 24/25 vaccinees in the Ty21a group. One volunteer had 5 ASC/106 PBMC to S. Paratyphi C (Fig. 1). On day 7, circulating ASC reactive with S. Paratyphi A, B and C-specific ASC were found in 23/25, 22/25 and 9/25 volunteers, respectively (Fig. 1). The magnitude (mean with 95% CI) and statistical comparison between the responses to the various strains are shown in Table 1. The immunoglobulin isotype distribution of the ASC responses in the Ty21a group has been shown in our previous report [11]: the responses were mostly co-dominated by IgA and IgM. Comparison of the ASC responses between Vi and Ty21a groups
The mean and 95% CI of the ASC responses to S. Typhi, Paratyphi A, B, C and Yersinia enterocolitica in the Vivaccinated group are presented in Table 1 together with the comparison of responses to the various strains. Comparisons between the Vi and Ty21a groups are shown in Fig. 3. The numbers of plasmablast reactive with S. Paratyphi A and B proved significantly lower in the Vi than the Ty21a group, while those reactive with S. Paratyphi C were higher in the Vi group.
Discussion In a situation where the incidence of paratyphoid fever is increasing along with an increasing antimicrobial resistance, and no vaccines against paratyphoid fever are available for clinical use, it is imperative to explore whether any of the current typhoid vaccines could be of help by affording cross-protection against the disease. Some reports suggest clinical efficacy by the oral whole-cell Ty21a vaccine [12, 13] or by the old parenteral whole-cell vaccine TAB (no longer in use) [32], and others provide consistent evidence of cross-reactive immune responses against paratyphoid strains by the Ty21a vaccine [11, 14– 17]. In contrast, some reports have found no crossprotection with Ty21a [33] or TAB [34], or immunological reactivity with TAB against Paratyphi A [15, 16]. We only know of two prospective clinical reports [5, 35] and one retrospective [13] evaluation of the potential cross-protective efficacy, and no immunological studies measuring the cross-reactivity of the Vi vaccine against paratyphoid
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Figure 1 Cross-reactive plasmablast response to Salmonella Paratyphi strains before and after immunization with typhoid vaccines. Numbers of circulating antigen-specific plasmablasts (identified as antibody-secreting cells, ASC) to S. Paratyphi A, S. Paratyphi B and S. Paratyphi C in 25 volunteers vaccinated with the Vi polysaccharide and 25 with the Ty21a vaccine. The lines represent the results of individual vaccinees in an assay for antigen-specific Ig (A+G+M) plasmablasts on days 0 and 7 after vaccination. LOD = limit of detection (of the response).
strains. The present study shows that this vaccine induces cross-reactive responses to all paratyphoidal strains, yet the responses mostly remain very low. The cross-reactive response seen to S. Paratyphi A, B and C deserves to be expounded on. Our observation propounds the idea that these strains and the Vi vaccine carry antigenic structures in common. Indeed, S. Paratyphi C carries the Vi antigen, which accounts for the crossreactivity found after Vi vaccination. S. Paratyphi A and B, by contrast, are known to be Vi-negative and thus unlikely to produce cross-reactivity – not unless the Vi vaccine preparation also contains other, cross-reactive structures. The O-12 antigen offers the most plausible explanation, as we have recently reported that the Vi vaccine elicits an antibody response to O-9,12 [19]. Notably, as the Vi Ó 2014 John Wiley & Sons Ltd
antigen in the vaccine has originally been derived from the wild-type S. Typhi Ty2, traces of cell wall components are still left in the preparation after purification [19, 36]. The human immune system simply appears sensitive enough to recognize such minimal amounts and to elicit a measurable immune response against these structures. The plasmablast responses against S. Paratyphi A and B elicited by the Vi vaccine were found to be significantly lower than that elicited by the Ty21a vaccine or that found earlier in patients with typhoid fever [11] (mean responses to S. Paratyphi A 20 versus 114 ASC/106 PBMC and to B 22 versus 137 in the Vi and Ty21a groups, respectively). In both groups, the responses are considered to be targeted against the O-12 antigen shared between them. The significantly higher cross-reactive response in the Ty21a
226 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. Table 1 Plasmablast responses to S. Typhi, Paratyphi A/B/C and Yersinia enterocolitica and results of statistical comparisons. Plasmablast response Vaccination group/ Bacterial strain Vi group S. Typhi S. Paratyphi A S. Paratyphi B S. Paratyphi C Yersinia enterocolitica Ty21a group S. Typhi S. Paratyphi A S. Paratyphi B S. Paratyphi C Yersinia enterocolitica
Mean
95% CI
Comparison with Percentage of response to S. Typhi
S. Typhi
S. Paratyphi A
S. Paratyphi B
S. Paratyphi C
149 20 22 22 2
1–217 2–37 4–39 8–35 1–3
13 15 15 1
*** *** *** ***
NS NS **
NS ***
***
339 114 137 4 0
155–521 31–197 39–236 1–6 0–1
34 40 1 0
*** *** *** ***
NS ** ***
*** ***
***
ns, not significant. Numbers of plasmablasts (ASC) specific to various antigens/bacterial strains (S. Typhi = Typhoidal O antigens O-9,12 + Vi antigen + H-d; S. Paratyphi A, B, C and Yersinia enterocolitica) in 25 volunteers vaccinated 1 week earlier with the oral Vi polysaccharide or Ty21a vaccine (means and 95% confidence intervals); magnitude of the response to various bacterial strains in percentages of the S. Typhi-specific response and statistical comparison (Bonferroni corrected Student’s t-test of logarithmically transformed data) between the responses to various strains. Significant differences are indicated with asterisks (***P < 0.001; **0.001 < P < 0.01). The data of ASC specific to S. Typhi and the negative control strain, Yersinia enterocolitica, were retrieved from our previous report [23].
Figure 2 Immunoglobulin isotype distribution of S. Paratyphoid-specific plasmablasts in volunteers vaccinated with the Vi polysaccharide vaccine. Immunoglobulin isotype distribution of antibodies secreted by plasmablast reactive with S. Paratyphi A/B/C (ASC/106 PBMC) in 25 volunteers immunized with the Vi polysaccharide vaccine. The dots represent results of individual vaccinees, and the lines the means of the numbers of ASC of the IgA, IgG or IgM isotype 7 days after vaccination.
group accords with the O-9,12-specific response being higher in the Ty21a than in the Vi group in our recent study [19]. This can be seen as a logical finding also from the perspective that O antigen is an essential part or the Ty21a vaccine, while in the Vi vaccine, it only represents a contamination remaining after the purification process. It should be pointed out that there is some batch-to-batch variation in the LPS contents of the Vi vaccines which may influence the magnitude of the ensuing cross-reactive immune responses. As expected, the Vi vaccine was more potent than the Ty21a vaccine in inducing a cross-reactive response against S. Paratyphi C; this pathogen carries the Vi antigen not presented by the Ty21a vaccine. Notably, we showed recently that the ELISPOT assay is more sensitive when using a purified Vi antigen instead of whole-cell S. Typhi
for coating. Similarly, the cross-reactive response to S. Paratyphi C detected with the present assay appeared lower than the Vi-specific response of the same volunteers in our recent study exploring humoral responses to various typhoidal antigens [19]. From the perspective of using the Vi vaccine for protection against paratyphoid fever, the crucial question remains whether the low cross-reactive responses have clinical significance. The plasmablasts have been suggested to serve as surrogate markers for protection [11], as their numbers increase together with protective efficacy against typhoid fever in field trials using the same regimens [21, 37–39]. This parallel increase was questioned in a recent study by Wahid et al. [17]; they considered that the significant cross-reactive plasmablast response to S. Paratyphi A in Ty21a-vaccinated volunteers was not in
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Figure 3 Comparison of the cross-reactive plasmablast responses between volunteers in the Vi or the Ty21a groups. Numbers of circulating S. Paratyphi A, B and C-specific plasmablasts (ASC) in 25 volunteers vaccinated with Vi polysaccharide and 25 with the S. Typhi Ty21a vaccine. The dots represent the results of individual volunteers, and lines the means of the numbers of Ig(A+G+M) ASC 7 days after vaccination. The statistical comparisons (Student’s t-test of logarithmically transformed data) between the groups are indicated with asterisks (*P < 0.05). LOD = limit of detection (of the response).
line with a large field trial in Plaju, Indonesia [33], where the same vaccine was found not to afford protection against the pathogen. However, in Plaju, the vaccine doses were given a whole week apart, not at a two-day interval as recommended by the manufacturer. When the schedule is extended, the effective dose is likely to become significantly reduced, for an active intestinal immunity developing within 1 week [21, 28] will probably act against the subsequent vaccine doses [40, 41]. Hence, in Plaju, the actual vaccinations may not have amounted to much more than one dose. This might also account for the exceptionally low level of efficacy against typhoid fever (42%) recorded in that study [33]. Even if the numbers of crossreactive plasmablasts have not been explored with an extended dosing schedule, such low-efficacy figures against typhoid fever is likely to signify only low cross-protection against paratyphoid strains, if any. Even if the numbers of plasmablasts appear to correlate with the level of protective efficacy [21, 37–39], some caution should be taken in interpreting study results, and further studies should, of course, be carried out to validate this approach. Accordingly, a comparison between protection shown in field trials and the number of plasmablasts in the present study would be of interest here. Levine et al. [12] reported a protective efficacy of 49% for Ty21a against S. Paratyphi B; and in our assay using the same regimen and dosing, the geometric mean of the responses to S. Paratyphi B was 51 ASC/106 PBMC. If the plasmablasts could be used as surrogate markers of protection, our results would suggest that the cross-reactive response to the Vi vaccine be too low (geometric mean 11 ASC/106 PBMC against S. Paratyphi B) to indicate cross-protection against
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paratyphoid fever. The insufficient responses in the Vi group accord with results from field trials: Yang et al. [35] reported no protection with Vi vaccine against S. Paratyphi A in a large field trial comprising 131 000 volunteers in China. Meltzer et al. [13] showed an increase in paratyphoid cases among Israeli travellers when switching over to the Vi from the Ty21a vaccine, and Dong et al. [5] found an increase in paratyphoid A cases after the introduction of the Vi vaccine in Guangxi, China. To our knowledge, no studies have assessed the protective efficacy of the Vi vaccine against S. Paratyphi B or C. It is noteworthy that even if the cross-reactive responses in the Vi group were low in general, some individual volunteers had higher responses. Based on the figures on protection and geometric means of ASC presented above, we set an arbitrary limit of 50 ASC/106 PBMC to see how many in the two vaccination groups would reach it. In the Vi group, 8%, 12% and 16%, and in the Ty21a group, 39%, 65% and 0% had more than 50 ASC/106 PBMC against S. Paratyphi A, B and C, respectively. Should plasmablast numbers act as a surrogate marker for protection, our data would imply that in some individual cases also the Vi vaccine could elicit a cross-protective immune response against paratyphoid fever. As to S. Paratyphi C, the clinical significance of any response is limited as long as the pathogen remains such a rare cause of paratyphoid fever. The immunoglobulin isotype distribution of the responses against paratyphoidal strains showed a predominance of IgA- and IgM-ASC in the Ty21a group [11], consistent with previous studies on oral immunization [21, 28, 42]. In the Vi group, the three isotypes were found equally presented, which accords with the fact that while
228 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. parenteral protein vaccines elicit a predominantly IgG-dominated response [43], parenteral polysaccharide vaccines also induce a significant IgA response [28, 44]. Studies of non-typhoidal Salmonella strains suggest that O antigen-specific antibodies may contribute to protection. MacLennan et al. [45] have shown in African children that O antigen-specific serum antibodies have a protective role in antibody-induced complement-mediated killing of nontyphoid Salmonellae (NTS). They have also found a rise in anti-Salmonella IgG and IgM antibody titres and serum bactericidal activity with age, corresponding with a fall in bacteremic NTS cases [45], suggesting that serum Salmonella-specific antibodies may serve to protect against invasive salmonellosis. In addition, both intestinal [46– 48] and systemic [49, 50] O antigen-specific antibodies have proven protective against salmonellosis in numerous animal experiments. To conclude, the Vi vaccine elicits a cross-reactive plasmablast response to all three paratyphoid strains. The responses to S. Paratyphi A and B, which do not carry the Vi antigen, are most likely to be caused by traces of cell wall polysaccharides remaining in the vaccine preparation after purification. In the majority of volunteers, the crossreactive responses remain very low, suggesting only limited clinical significance.
Acknowledgment We thank Tuomo Kauko, Department of Biostatistics, University of Turku, Finland, for help with the statistical analyses and Professor Anja Siitonen, Bacteriology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare Finland, for providing the S. Typhi, S. Paratyphi A and B strains for our assays. This work was supported by Crucell Switzerland AG, the Finnish Governmental Subsidy for Health Science Research and the Finnish Concordia Fund (SP) and Biomedicum Helsinki Foundation (SP). The funding sources had no involvement in study design, data collection, analysis, interpretation of data, writing of the report, and the decision to submit the article for publication.
Contributions AK, JK and SP conceived and designed the experiments, SP carried them out, SP and JK analysed the data. AK contributed reagents, materials and analysis equipment. SP, JK and AK wrote the report.
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37 Kantele A. Peripheral blood antibody-secreting cells in the evaluation of the immune response to an oral vaccine. J Biotechnol 1996;44:217–24. 38 Ferreccio C, Levine MM, Rodriguez H, Contreras R. Comparative efficacy of two, three, or four doses of TY21a live oral typhoid vaccine in enteric-coated capsules: a field trial in an endemic area. J Infect Dis 1989;159:766–9. 39 Levine MM, Ferreccio C, Black RE, Germanier R. Large-scale field trial of Ty21a live oral typhoid vaccine in enteric-coated capsule formulation. Lancet 1987;1:1049–52. 40 Kantele A, Kantele JM, Arvilommi H, M€akel€a PH. Active immunity is seen as a reduction in the cell response to oral live vaccine. Vaccine 1991;9:428–31. 41 Forrest BD. Impairment of immunogenicity of Salmonella typhi Ty21a due to preexisting cross-reacting intestinal antibodies. J Infect Dis 1992;166:210–2. 42 Lycke N, Lindholm L, Holmgren J. Cholera antibody production in vitro by peripheral blood lymphocytes following oral immunization of humans and mice. Clin Exp Immunol 1985;62:39–47. 43 Kantele A, Savilahti E, Tiimonen H, Iikkanen K, Autio S, Kantele JM. Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter. Eur J Immunol 2003;33:3275–83. 44 Lue C, Tarkowski A, Mestecky J. Systemic immunization with pneumococcal polysaccharide vaccine induces a predominant IgA response of peripheral blood lymphocytes and increases of both serum and secretory anti-pneumococcal antibodies. J Immunol 1988;140:3793–800. 45 MacLennan CA, Gondwe EN, Msefula CL et al. The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. J Clin Invest 2008;118:1553–62. 46 Michetti P, Mahan MJ, Slauch JM, Mekalanos JJ, Neutra MR. Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium. Infect Immun 1992;60:1786–92. 47 Michetti P, Porta N, Mahan MJ et al. Monoclonal immunoglobulin A prevents adherence and invasion of polarized epithelial cell monolayers by Salmonella typhimurium. Gastroenterology 1994;107:915–23. 48 Endt K, Stecher B, Chaffron S et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog 2010;6:e1001097. 49 Saxen H, M€akel€a O, Svenson SB. Isotype of protective anti-Salmonella antibodies in experimental mouse salmonellosis. Infect Immun 1984;44:633–6. 50 Saxen H. Mechanism of the protective action of anti-Salmonella IgM in experimental mouse salmonellosis. J Gen Microbiol 1984;130:2277–83.
Cross-Reactive Immune Response Induced by the Vi Capsular Polysaccharide Typhoid Vaccine Against Salmonella Paratyphi Strains S. H. Pakkanen*†, J. M. Kantele‡ & A. Kantele*†§
Abstract *Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; †Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland; ‡Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland; and §Department of Medicine, Institute of Clinical Medicine, University of Helsinki, Helsinki, Finland
Received 16 November 2013; Accepted in revised form 22 December 2013 Correspondence to: Adjunct Professor Anu Kantele, Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, POB 348, FIN-00029 HUS Helsinki, Finland. E-mail: [email protected]
There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross-reactive immune response against S. Paratyphi A and B, and some reports suggest cross-protective efficacy against the disease. These findings are ascribed to the O-12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O-9,12. Twenty-five volunteers immunized with the parenteral Vi vaccine (Typherixâ) were explored for plasmablasts cross-reactive with paratyphoid strains; the responses were compared to those in 25 age- and gender-matched volunteers immunized with Ty21a (Vivotifâ). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi- and Ty21a-vaccinated volunteers had circulating plasmablasts producing antibodies cross-reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross-reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi-negative, the result can be explained by trace amounts of bacterial cell wall O-12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined.
Introduction There is an urgent need for vaccines against paratyphoid fever, as both the incidence of the disease [1–5] and antimicrobial resistance are increasing [1, 3, 6–9] and there are no vaccines available. Paratyphoid fever is caused by Salmonella enterica subsp. enterica serovar Paratyphi A and B (S. Paratyphi A and B) and, rarely, Salmonella enterica subsp. enterica serovar Paratyphi C (S. Paratyphi C). The global number of paratyphoid fever cases has been estimated at 5.4 million cases per year [10]. Recently, it has been proposed that the oral live attenuated Ty21a typhoid vaccine (Vivotifâ) could be used as a surrogate vaccine against paratyphoid fever until a vaccine specifically targeted against this disease becomes available [11]. This line of thinking is based on reports of Ty21a vaccine conferring cross-protective efficacy against paratyphoid fever [12, 13] and, consistent with them, on immunological studies
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attesting that Ty21a-vaccinated volunteers develop substantial cross-reactive cell- [14–16] and antibody-mediated [11, 17] immune responses against S. Paratyphi A and B. Cross-protection [12] and cross-reactive immune response [11, 14, 17] elicited by the Ty21a vaccine have been explained by the common O-12 antigen found both on S. Typhi, Ty21a vaccine strain, and on S. Paratyphi A and B. Accordingly, the Ty21a has been shown to elicit a significant plasmablast response to all these strains, but not against S. Paratyphi C and S. Egusi, which carry unrelated O antigen types (O-6,7 and O-41, respectively) [11]. The Ty21a and the Vi capsular polysaccharide vaccines have different antigenic compositions [7, 18]. The Ty21a is a whole-cell vaccine sharing the O antigens 9 and 12 with S. Typhi, but lacking the Vi polysaccharide, whereas the Vi vaccine is a purified polysaccharide preparation that has been considered to share only the Vi antigen with S. Typhi. While the Ty21a vaccine elicits a significant cross-reactive
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immune response solely against S. Paratyphi A and B [11], the Vi vaccine is expected to induce a response only to S. Paratyphi C which, in contrast to S. Paratyphi A and B, carries the Vi antigen. However, as a preparation initially isolated from the S. Typhi Ty2 strain, the Vi vaccine contains traces of contaminating typhoid lipopolysaccharide with O-9,12 antigens. In our recent study, we demonstrated that this amount suffices to elicit an immune response [19]. This was found by exploring plasmablasts appearing in the circulation after vaccination. They represent maturing B cells trafficking from the initial site of antigen encounter via blood to sites where they settle down as plasma cells [20]. Assessing circulating plasmablasts has proved to be an especially sensitive approach to evaluating the humoral response to vaccinations, either by oral [19, 21–28] or parenteral route [19, 22, 24, 27, 28]. The recent observation of O antigen-specific humoral immune response induced by the Vi vaccine raises a question of whether this vaccine could elicit a cross-reactive immune response against S. Paratyphi A and B, consistently with the Ty21a vaccine.
Material and methods Ethics statement. The study was conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved both by the ethics committee of the Helsinki University Central Hospital and the Finnish Medicines Agency and entered in the registry of Current Controlled Trials Ltd. c/o BioMed Central (International Standard Randomized Controlled Trial Number ISRCTN68125331). Written informed consent was obtained from all study subjects. Study design. Cross-reactive immune response to S. Paratyphi strains was explored in volunteers vaccinated with Vi polysaccharide (Typherixâ; GlaxoSmithKline Biologicals s.a., Rixensart, Belgium) and compared to that in age- and gender-matched volunteers vaccinated with the live oral typhoid S. Typhi Ty21a (Vivotifâ; Crucell, Leiden, Netherlands) vaccine. Circulating plasmablasts specific to S. Paratyphi A and B (O-12 antigen in common with S. Typhi) and S. Paratyphi C (Vi antigen in common with S. Typhi) were studied by enzyme-linked immunospot assay (ELISPOT) before and 7 days after administering the Vi vaccine or the first dose of Ty21a to identify cross-reactive antibody-secreting cells (ASC). For comparison, data on immune responses to the various typhoid antigens and Yersinia enterocolitica (negative control) in both of these vaccination groups were retrieved from our recent report [19], and responses to paratyphoid strains were compared to these. Results of the paratyphoid-specific responses in 17/25 volunteers in the Ty21a group have been incorporated in our recently published data, where no age- and gender-matched controls were included [11]. The present study only included age- and gender-matched
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volunteers who had initially been randomized to receive either the Ty21a or the parenteral Vi vaccine. The study was conducted at the Helsinki University Central Hospital and at the Haartman Institute, University of Helsinki between December 2009 and October 2010. Volunteers, vaccinations and samples. Fifty healthy volunteers were randomized into two age- and gender-matched groups of 25 volunteers (both groups consisted of 17 females and 8 males aged between 22 and 62; average 32 years) with no history of typhoid vaccination or typhoid or paratyphoid fever, the numbers of volunteers were evaluated in prestudy power calculations based on responses in pilot experiments. A parenteral Vi polysaccharide vaccine (Typherixâ; GlaxoSmithKline Biologicals s.a., lots ATYPB084BC and ATYPB096AF) was given to one group, and a live oral vaccine containing ≥2 9 109 live Salmonella Typhi Ty21a bacteria (Vivotifâ, Crucell, lot 3001777) to the other. The amount of contaminating endotoxin in the Typherix vaccine preparation was 13.30 EU per dose for lot ATYPB096AF and 27.00 EU per dose for lot ATYPB084BC (data provided by the manufacturer). The parenteral vaccine was administered with a 25-mmlength needle as one 0.5 ml dose in the left deltoid muscle of the volunteers on day 0. The oral vaccine was administered as one enteric-coated capsule on days 0, 2 and 4, following the manufacturer’s recommendation. The vaccine type given to the first volunteer of each age was determined by raffle, and the second, age- and gendermatched subject, was given the other vaccine type. Notably, the results of the assays on the coded ELISPOT plates were read by a blinded person with no knowledge about the types of vaccine given in the various cases or which plates represented age- and gender-matched pairs. Both vaccines proved well tolerated, as reported in our previous study where all adverse effects are described [19]. There were no dropouts in the study. A sample of peripheral venous blood was drawn on days 0 and 7 after administering the Vi vaccine or the first dose of Ty21a. Previous studies of mucosal [21, 28, 29] and parenteral [28] antigen administration show that the numbers of ASC peak on day 7 after antigen encounter, and the cells disappear by day 14–16. Accordingly, day 7 was chosen as the time point for catching circulating ASC. Antigen. Strains of S. Paratyphi A, B and C were used for coating in the ELISPOT assay. The results were compared to data retrieved from our previous study exploring in the same volunteers the response against S. Typhi and Yersinia enterocolitica (negative control). The total response to S. Typhi had been calculated by adding together the response to typhoidal O-9,12 antigen (represented by Salmonella SL2404 strain), Vi antigen and H-d (represented by S. Egusi) [19]. The S. Paratyphi A (RHS6716) and B (RHS6744) strains were obtained from the collection of the National Institute for Health and Welfare (THL), Helsinki, Finland,
224 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. and the S. Paratyphi C strain from the ACTT (ATCC13428; American Type Culture Collection, Manassas, VA, USA). To confirm the expressions of the O and Vi antigens, each bacterial strain was analysed by the Finnish national reference laboratorium of the THL according to their routines. The preparation of the antigen has been explained in detail previously [11, 21]. ELISPOT assay of specific ASC. PBMC were separated using Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) centrifugation of fresh heparinized venous blood [21]. These cells were explored with ELISPOT for ASC specific to the various paratyphoid strains, as described previously [15]. In brief, 96-well microtitre plate wells (Maxisorp, Nunc, Roskilde, Denmark) were coated with a whole-cell preparation of formalin-killed bacteria. The PBMC were incubated in the wells for 2 h, and antibodies secreted during this time were detected with alkaline phosphataseconjugated goat anti-human IgA (Sigma-Aldrich, St. Louis, MO, USA), IgG (Sigma-Aldrich) and IgM (SouthernBiotech, Birmingham, England). The substrate (bromo-4chloro-3-indolyl phosphate p-toluidine salt; Sigma-Aldrich) was added in melted agarose. The spots were enumerated with AID Elispot reader, and each spot was interpreted as a print of one ASC. The specificity, linearity, stability and intermediate precision of the ELISPOT assay have all been validated (data not shown). A responder was defined as a person with at least 3 IgA+IgG + IgM-ASC/106 PBMC and marked as LOD (limit of detection) of the response in the figures. This limit has been determined over the assay validation process. Statistics. Statistical analyses were carried out with JMP software version 9.0.0 (SAS Institute Inc, Cary, NC, USA). After logarithmic transformation of the data, the distributions of the numbers of ASC were tested with Shapiro– Wilk’s test. All proved normal. Student’s t-test was used for comparisons, applying the Bonferroni correction to various antigens. Differences were considered significant when P < 0.05. The results are given as the means and 95% confidence intervals (CI) for the number of ASC.
Results Antibody-secreting cells response in the Vi group
Before vaccination, no S. Paratyphi A/B or C-specific ASC were found in the circulation of 24/25 vaccinees in the Vi group (Fig. 1). One volunteer, who had 1 week earlier had an upper respiratory tract infection, had 98 ASC/106 PBMC to S. Paratyphi A, 95 to B and 60 to Paratyphi C (Fig. 1). We have previously shown that infections can be associated with a certain degree of polyclonal immune response [30, 31]. On day 7, circulating ASC reactive with S. Paratyphi A, B and C were found in 15/25, 18/25 and 16/25 volunteers
(Fig. 1). The magnitude (mean with 95% CI) and statistical comparisons between the responses to the various strains are shown in Table 1. IgA-, IgG- and IgM-ASC were equally presented in the cross-reactive responses in the Vi group (Fig. 2). Antibody-secreting cells response in the Ty21a group
Before vaccination, no S. Paratyphi A or B -specific ASC were found in the circulation of 24/25 vaccinees in the Ty21a group. One volunteer had 5 ASC/106 PBMC to S. Paratyphi C (Fig. 1). On day 7, circulating ASC reactive with S. Paratyphi A, B and C-specific ASC were found in 23/25, 22/25 and 9/25 volunteers, respectively (Fig. 1). The magnitude (mean with 95% CI) and statistical comparison between the responses to the various strains are shown in Table 1. The immunoglobulin isotype distribution of the ASC responses in the Ty21a group has been shown in our previous report [11]: the responses were mostly co-dominated by IgA and IgM. Comparison of the ASC responses between Vi and Ty21a groups
The mean and 95% CI of the ASC responses to S. Typhi, Paratyphi A, B, C and Yersinia enterocolitica in the Vivaccinated group are presented in Table 1 together with the comparison of responses to the various strains. Comparisons between the Vi and Ty21a groups are shown in Fig. 3. The numbers of plasmablast reactive with S. Paratyphi A and B proved significantly lower in the Vi than the Ty21a group, while those reactive with S. Paratyphi C were higher in the Vi group.
Discussion In a situation where the incidence of paratyphoid fever is increasing along with an increasing antimicrobial resistance, and no vaccines against paratyphoid fever are available for clinical use, it is imperative to explore whether any of the current typhoid vaccines could be of help by affording cross-protection against the disease. Some reports suggest clinical efficacy by the oral whole-cell Ty21a vaccine [12, 13] or by the old parenteral whole-cell vaccine TAB (no longer in use) [32], and others provide consistent evidence of cross-reactive immune responses against paratyphoid strains by the Ty21a vaccine [11, 14– 17]. In contrast, some reports have found no crossprotection with Ty21a [33] or TAB [34], or immunological reactivity with TAB against Paratyphi A [15, 16]. We only know of two prospective clinical reports [5, 35] and one retrospective [13] evaluation of the potential cross-protective efficacy, and no immunological studies measuring the cross-reactivity of the Vi vaccine against paratyphoid
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Figure 1 Cross-reactive plasmablast response to Salmonella Paratyphi strains before and after immunization with typhoid vaccines. Numbers of circulating antigen-specific plasmablasts (identified as antibody-secreting cells, ASC) to S. Paratyphi A, S. Paratyphi B and S. Paratyphi C in 25 volunteers vaccinated with the Vi polysaccharide and 25 with the Ty21a vaccine. The lines represent the results of individual vaccinees in an assay for antigen-specific Ig (A+G+M) plasmablasts on days 0 and 7 after vaccination. LOD = limit of detection (of the response).
strains. The present study shows that this vaccine induces cross-reactive responses to all paratyphoidal strains, yet the responses mostly remain very low. The cross-reactive response seen to S. Paratyphi A, B and C deserves to be expounded on. Our observation propounds the idea that these strains and the Vi vaccine carry antigenic structures in common. Indeed, S. Paratyphi C carries the Vi antigen, which accounts for the crossreactivity found after Vi vaccination. S. Paratyphi A and B, by contrast, are known to be Vi-negative and thus unlikely to produce cross-reactivity – not unless the Vi vaccine preparation also contains other, cross-reactive structures. The O-12 antigen offers the most plausible explanation, as we have recently reported that the Vi vaccine elicits an antibody response to O-9,12 [19]. Notably, as the Vi Ó 2014 John Wiley & Sons Ltd
antigen in the vaccine has originally been derived from the wild-type S. Typhi Ty2, traces of cell wall components are still left in the preparation after purification [19, 36]. The human immune system simply appears sensitive enough to recognize such minimal amounts and to elicit a measurable immune response against these structures. The plasmablast responses against S. Paratyphi A and B elicited by the Vi vaccine were found to be significantly lower than that elicited by the Ty21a vaccine or that found earlier in patients with typhoid fever [11] (mean responses to S. Paratyphi A 20 versus 114 ASC/106 PBMC and to B 22 versus 137 in the Vi and Ty21a groups, respectively). In both groups, the responses are considered to be targeted against the O-12 antigen shared between them. The significantly higher cross-reactive response in the Ty21a
226 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. Table 1 Plasmablast responses to S. Typhi, Paratyphi A/B/C and Yersinia enterocolitica and results of statistical comparisons. Plasmablast response Vaccination group/ Bacterial strain Vi group S. Typhi S. Paratyphi A S. Paratyphi B S. Paratyphi C Yersinia enterocolitica Ty21a group S. Typhi S. Paratyphi A S. Paratyphi B S. Paratyphi C Yersinia enterocolitica
Mean
95% CI
Comparison with Percentage of response to S. Typhi
S. Typhi
S. Paratyphi A
S. Paratyphi B
S. Paratyphi C
149 20 22 22 2
1–217 2–37 4–39 8–35 1–3
13 15 15 1
*** *** *** ***
NS NS **
NS ***
***
339 114 137 4 0
155–521 31–197 39–236 1–6 0–1
34 40 1 0
*** *** *** ***
NS ** ***
*** ***
***
ns, not significant. Numbers of plasmablasts (ASC) specific to various antigens/bacterial strains (S. Typhi = Typhoidal O antigens O-9,12 + Vi antigen + H-d; S. Paratyphi A, B, C and Yersinia enterocolitica) in 25 volunteers vaccinated 1 week earlier with the oral Vi polysaccharide or Ty21a vaccine (means and 95% confidence intervals); magnitude of the response to various bacterial strains in percentages of the S. Typhi-specific response and statistical comparison (Bonferroni corrected Student’s t-test of logarithmically transformed data) between the responses to various strains. Significant differences are indicated with asterisks (***P < 0.001; **0.001 < P < 0.01). The data of ASC specific to S. Typhi and the negative control strain, Yersinia enterocolitica, were retrieved from our previous report [23].
Figure 2 Immunoglobulin isotype distribution of S. Paratyphoid-specific plasmablasts in volunteers vaccinated with the Vi polysaccharide vaccine. Immunoglobulin isotype distribution of antibodies secreted by plasmablast reactive with S. Paratyphi A/B/C (ASC/106 PBMC) in 25 volunteers immunized with the Vi polysaccharide vaccine. The dots represent results of individual vaccinees, and the lines the means of the numbers of ASC of the IgA, IgG or IgM isotype 7 days after vaccination.
group accords with the O-9,12-specific response being higher in the Ty21a than in the Vi group in our recent study [19]. This can be seen as a logical finding also from the perspective that O antigen is an essential part or the Ty21a vaccine, while in the Vi vaccine, it only represents a contamination remaining after the purification process. It should be pointed out that there is some batch-to-batch variation in the LPS contents of the Vi vaccines which may influence the magnitude of the ensuing cross-reactive immune responses. As expected, the Vi vaccine was more potent than the Ty21a vaccine in inducing a cross-reactive response against S. Paratyphi C; this pathogen carries the Vi antigen not presented by the Ty21a vaccine. Notably, we showed recently that the ELISPOT assay is more sensitive when using a purified Vi antigen instead of whole-cell S. Typhi
for coating. Similarly, the cross-reactive response to S. Paratyphi C detected with the present assay appeared lower than the Vi-specific response of the same volunteers in our recent study exploring humoral responses to various typhoidal antigens [19]. From the perspective of using the Vi vaccine for protection against paratyphoid fever, the crucial question remains whether the low cross-reactive responses have clinical significance. The plasmablasts have been suggested to serve as surrogate markers for protection [11], as their numbers increase together with protective efficacy against typhoid fever in field trials using the same regimens [21, 37–39]. This parallel increase was questioned in a recent study by Wahid et al. [17]; they considered that the significant cross-reactive plasmablast response to S. Paratyphi A in Ty21a-vaccinated volunteers was not in
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Figure 3 Comparison of the cross-reactive plasmablast responses between volunteers in the Vi or the Ty21a groups. Numbers of circulating S. Paratyphi A, B and C-specific plasmablasts (ASC) in 25 volunteers vaccinated with Vi polysaccharide and 25 with the S. Typhi Ty21a vaccine. The dots represent the results of individual volunteers, and lines the means of the numbers of Ig(A+G+M) ASC 7 days after vaccination. The statistical comparisons (Student’s t-test of logarithmically transformed data) between the groups are indicated with asterisks (*P < 0.05). LOD = limit of detection (of the response).
line with a large field trial in Plaju, Indonesia [33], where the same vaccine was found not to afford protection against the pathogen. However, in Plaju, the vaccine doses were given a whole week apart, not at a two-day interval as recommended by the manufacturer. When the schedule is extended, the effective dose is likely to become significantly reduced, for an active intestinal immunity developing within 1 week [21, 28] will probably act against the subsequent vaccine doses [40, 41]. Hence, in Plaju, the actual vaccinations may not have amounted to much more than one dose. This might also account for the exceptionally low level of efficacy against typhoid fever (42%) recorded in that study [33]. Even if the numbers of crossreactive plasmablasts have not been explored with an extended dosing schedule, such low-efficacy figures against typhoid fever is likely to signify only low cross-protection against paratyphoid strains, if any. Even if the numbers of plasmablasts appear to correlate with the level of protective efficacy [21, 37–39], some caution should be taken in interpreting study results, and further studies should, of course, be carried out to validate this approach. Accordingly, a comparison between protection shown in field trials and the number of plasmablasts in the present study would be of interest here. Levine et al. [12] reported a protective efficacy of 49% for Ty21a against S. Paratyphi B; and in our assay using the same regimen and dosing, the geometric mean of the responses to S. Paratyphi B was 51 ASC/106 PBMC. If the plasmablasts could be used as surrogate markers of protection, our results would suggest that the cross-reactive response to the Vi vaccine be too low (geometric mean 11 ASC/106 PBMC against S. Paratyphi B) to indicate cross-protection against
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paratyphoid fever. The insufficient responses in the Vi group accord with results from field trials: Yang et al. [35] reported no protection with Vi vaccine against S. Paratyphi A in a large field trial comprising 131 000 volunteers in China. Meltzer et al. [13] showed an increase in paratyphoid cases among Israeli travellers when switching over to the Vi from the Ty21a vaccine, and Dong et al. [5] found an increase in paratyphoid A cases after the introduction of the Vi vaccine in Guangxi, China. To our knowledge, no studies have assessed the protective efficacy of the Vi vaccine against S. Paratyphi B or C. It is noteworthy that even if the cross-reactive responses in the Vi group were low in general, some individual volunteers had higher responses. Based on the figures on protection and geometric means of ASC presented above, we set an arbitrary limit of 50 ASC/106 PBMC to see how many in the two vaccination groups would reach it. In the Vi group, 8%, 12% and 16%, and in the Ty21a group, 39%, 65% and 0% had more than 50 ASC/106 PBMC against S. Paratyphi A, B and C, respectively. Should plasmablast numbers act as a surrogate marker for protection, our data would imply that in some individual cases also the Vi vaccine could elicit a cross-protective immune response against paratyphoid fever. As to S. Paratyphi C, the clinical significance of any response is limited as long as the pathogen remains such a rare cause of paratyphoid fever. The immunoglobulin isotype distribution of the responses against paratyphoidal strains showed a predominance of IgA- and IgM-ASC in the Ty21a group [11], consistent with previous studies on oral immunization [21, 28, 42]. In the Vi group, the three isotypes were found equally presented, which accords with the fact that while
228 Cross-Reactivity to S. Paratyphi by Vi Vaccine S. H. Pakkanen et al. .................................................................................................................................................................. parenteral protein vaccines elicit a predominantly IgG-dominated response [43], parenteral polysaccharide vaccines also induce a significant IgA response [28, 44]. Studies of non-typhoidal Salmonella strains suggest that O antigen-specific antibodies may contribute to protection. MacLennan et al. [45] have shown in African children that O antigen-specific serum antibodies have a protective role in antibody-induced complement-mediated killing of nontyphoid Salmonellae (NTS). They have also found a rise in anti-Salmonella IgG and IgM antibody titres and serum bactericidal activity with age, corresponding with a fall in bacteremic NTS cases [45], suggesting that serum Salmonella-specific antibodies may serve to protect against invasive salmonellosis. In addition, both intestinal [46– 48] and systemic [49, 50] O antigen-specific antibodies have proven protective against salmonellosis in numerous animal experiments. To conclude, the Vi vaccine elicits a cross-reactive plasmablast response to all three paratyphoid strains. The responses to S. Paratyphi A and B, which do not carry the Vi antigen, are most likely to be caused by traces of cell wall polysaccharides remaining in the vaccine preparation after purification. In the majority of volunteers, the crossreactive responses remain very low, suggesting only limited clinical significance.
Acknowledgment We thank Tuomo Kauko, Department of Biostatistics, University of Turku, Finland, for help with the statistical analyses and Professor Anja Siitonen, Bacteriology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare Finland, for providing the S. Typhi, S. Paratyphi A and B strains for our assays. This work was supported by Crucell Switzerland AG, the Finnish Governmental Subsidy for Health Science Research and the Finnish Concordia Fund (SP) and Biomedicum Helsinki Foundation (SP). The funding sources had no involvement in study design, data collection, analysis, interpretation of data, writing of the report, and the decision to submit the article for publication.
Contributions AK, JK and SP conceived and designed the experiments, SP carried them out, SP and JK analysed the data. AK contributed reagents, materials and analysis equipment. SP, JK and AK wrote the report.
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