We studied salivary IgG and IgA antibody responses in PCV7 vaccinated and non-vaccinated children in a randomized controlled setting before nationwide PCV7 introduction in the Netherlands. We also studied the nasopharyngeal carriage of these children, which allowed us to study the impact of serotype-specific carriage on salivary antibody responses (for detailed carriage data see 
). We showed that in unvaccinated controls salivary anticapsular IgG and IgA antibodies were induced by natural boosting via carriage for most serotypes. Earlier it was already observed by Simell et al that natural exposure to pneumococci induced salivary IgA antibody responses 
. Our study confirms this observation, plus shows this is also true for boosting of salivary IgG. Until now, this association between salivary IgG antibodies and carriage was not observed, possibly because of the low salivary IgG levels which are commonly below the lower limit of detection when methods like EIA are used.
Systemic administration of 2 primary doses of PCV7 resulted in increased salivary IgG antibody responses against all vaccine serotypes compared to unvaccinated controls. An additional PCV7 booster dose at 11 months increased salivary IgG antibody levels one month later compared to 2 primary doses, illustrating that pneumococcal conjugate vaccines do contribute to salivary IgG antibody levels 
. For salivary IgA different dynamics in salivary antibody vaccine responses were observed. At 24 months of age the parallel increase in serotype-specific salivary IgA levels in the 2-dose and unvaccinated control group resulted in less pronounced differences between randomization groups, suggesting a parallel process of natural boosting. Our data even show that carriage-induced salivary IgA levels seems comparable to vaccine-induced IgA levels, which was most clear for serotype 19F. After PCV vaccinations, less vaccine serotype-specific carriage occurs 
. This might lead to less natural boosting of IgA anticapsular antibody levels compared to the control group, and can explain why also most previous studies do not observe a difference in salivary IgA levels between PCV7 vaccinated and unvaccinated children 
No significant differences were observed in non-vaccine serotype salivary antibody responses between controls and vaccinees, which is in correspondence with earlier reports 
. Interestingly, the increases in antibody levels against non-vaccine serotypes between 12 and 24 months of age, especially in the 2+1-dose group, most probably represent natural boosting. Although there is potentially more carriage of non-vaccine serotypes in the vaccinated groups, none of these tested serotypes were frequently encountered in conventional cultures in our carriage study 
. This might be due to large intervals between samples, however, polyreactivity on the same antigenic stimulus can not be ruled out 
, as well as cross-reactivity with other bacteria 
The exact contribution of PCV7 induced salivary IgA and IgG antibody levels in protection against nasopharyngeal carriage and disease is not yet known. We reported a 58% and 60% reduction in vaccine serotype pneumococcal carriage at the age of 24 months in both the 2-dose and 2+1-dose group compared to unvaccinated controls, respectively 
. In the present study however at this age no difference in salivary IgA antibody levels between the 2-dose and controls could be observed for most vaccine serotypes. This in contrast to significantly higher salivary IgG antibodies antibodies observed in vaccinated children where, in addition, a stepwise increase was observed between the control, 2-dose and 2+1-dose groups. This may suggest that vaccine-induced anti-capsular salivary IgG antibodies have a stronger contribution to protection against pneumococcal colonization then IgA. However, one has to realize that level of antibodies may not represent functionality 
and IgA antibodies have been shown to support anti-capsular complement-dependent opsonophagocytosis, and agglutination of the pneumococcus at the mucosal surface; functions which are not tested with our quantitative assay 
Lastly, we studied the potential correlation between serum and saliva antibodies in a small subgroup of children of which both saliva- and serum-samples were available. Although the small sample size does not allow for firm conclusions, we observed that after PCV7 administration salivary IgG antibody levels correlated well with serum antibody levels, supporting the hypothesis of IgG transport to the mucosal site 
. In PCV7 vaccinated children systemic IgG levels proved to be 10–20 fold higher than salivary IgG levels, in contrast to the IgA levels. For IgA the serum/saliva-ratio's are suggestive for a predominance of local production of IgA at the mucosal site. Especially for serotype 19F where higher salivary IgA levels were observed compared with serum levels supports this hypothesis. Also we were able to show both IgG and IgA salivary antibody levels increased with serotype 19F carriage.
The fact that serum and salivary IgG correlate well after PCV7 vaccination immediately raises two new questions: the first. whether this method could potentially be used as surveillance strategy; collection of saliva is less invasive, easy to obtain in sufficient quantities, and methods for antibody analysis are similar for saliva and serum. It might therefore be a reasonable alternative whenever invasive measurements are impossible. Second, whether measurement of salivary IgG levels might elicit a cut-off for protection against carriage. However, new studies with a larger cohort of participants should be executed to get a better insight into the answers to both questions.
This study was performed well before herd effects after PCV7 introduction in the Dutch national immunization program. In the coming years the impact of decreased vaccine serotype carriage on salivary antibody responses have to be evaluated, as natural boosting seems important in salivary antibody persistence. Salivary vaccine responses may therefore vary between regions and continents. Also primary doses at older ages, more doses or broader intervals between doses can impact vaccine immunogenicity, as well as other factors like concomitant childhood vaccinations or ethnic background variability 
. Some limitations of this study should be addressed. First, salivary IgA is susceptible to cleavage through bacterial IgA1 proteases 
. To prevent this, samples were immediately frozen after collection and analyzed directly after thawing. The randomized controlled setting also contributes to the reliability of the observed differences between groups. Second, due to the large 6-months interval of nasopharyngeal sampling, in between we will have missed carriage episodes in individuals. This might be the reason why not at all time points higher salivary antibody levels in colonized children could be observed compared with non-colonized children. Finally, the single colony method for serotyping may have resulted in missed multiple serotype carriage. Still for all of the 3 tested serotypes boosting of the immune system after natural exposure could be shown.
Strengths of our study include the randomized controlled study design with an unvaccinated control group. This made it possible to estimate the effect of PCV7 administration and pneumococcal carriage on salivary responses without the influences of temporal or geographical trends in distribution of circulating pneumococcal serotypes and to differentiate between true vaccine effects and natural immunity. Second, the highly sensitive multiplex technique (MIA) 
allowed to obverse less robust differences between the different time points and groups. Lastly IgA antibody levels strongly depend on the secretion flow rate of the participant during sample collection 
, in this study antibody levels were also corrected for total salivary protein, which did not change the results.
In conclusion, systemic administration of PCV7 proved to induce both salivary IgG and IgA antibodies. IgG antibody levels remained higher after 2+1-doses up till 24 months of age, while for IgA a strong increase was observed with age, independent of immunization. Nasopharyngeal carriage proved to be a major contributor to salivary antibody levels, especially IgA. We would like to advocate for new studies on salivary antibodies as potential immunological correlates of protection against pneumococcal carriage as well as on the potential usefulness of salivary antibodies in vaccine monitoring to further support this work.,.