This infant cohort in a low-resource tropical country, recruited before birth and followed up prospectively, provided a good opportunity to investigate potential differences between the effects of three BCG strains that are commonly used globally. We found significant differences in mycobacteria-specific and non-specific immune responses, and in the frequency of BCG-associated adverse events, according to the vaccine strain used. To our knowledge, this is the largest study to evaluate the effects of BCG strain on immune responses to the BCG vaccine and the only study to assess both specific and non-specific responses 
Other studies have shown that BCG elicits type 1 and type 2 responses, to both mycobacteria-specific and non-specific stimuli [28,29]
. Non-specific effects have also been reported at the clinical level, with reductions in non-TB infections and in overall mortality in BCG immunised infants [14,25–27,35,36]
. Furthermore, BCG has been shown to act non-specifically as a primer for other vaccines 
. Here we were able to conduct a broad analysis of the effect of BCG strain by comparing type 1 (IFN-γ), type 2 (IL-5 and IL-13) and regulatory (IL-10) responses to both mycobacteria-specific (cCFP and Ag85) and non-specific (TT and PHA) stimuli.
The results revealed three significant patterns of strain-dependent variability of immune responses to both mycobacteria-specific and non-specific stimuli: higher IFN-γ and IL-13 responses in the BCG-Denmark group; lower IL-5 responses in the BCG-Bulgaria group; and higher IL-10 responses in both the BCG-Denmark and BCG-Bulgaria group compared to BCG-Russia. Consistent with being at the greatest genetic distance from the other two strains 
, the cytokine responses of the BCG-Denmark group were the most divergent. Surprisingly however, they were also the highest overall, despite being most distantly related to the original M. bovis
. It is also interesting that BCG-Bulgaria and BCG-Russia behaved slightly differently in this cohort, despite being genetically identical, except for possible single nucleotide changes 
. As all infants were immunised with BCG, it is uncertain how these findings would relate to non-specific responses (such as the response to TT) amongst BCG-unvaccinated infants, however, differences between strains in non-specific effects were clearly demonstrated.
It is possible that the greater immunogenicity of BCG-Denmark may lead to better protection against TB. However, IFN-γ alone is an insufficient protective marker and it is feasible that higher regulatory IL-10 production in the same group may counteract its effects 
. The observation that IL-10 production differed between strains is contrary to a recent study 
that found that BCG did not stimulate an IL-10 response. This analysis suggests that the ability of BCG to stimulate an IL-10 response may be strain-dependent, although a study that compared BCG-Denmark to BCG-Brazil and BCG-Japan, found no such differences 
. Importantly, the differences across groups were observed in response to TT and PHA as well as to mycobacterial antigens, suggesting that the non-specific effects of BCG immunisation are likely to be dependent on the strain administered. The finding for TT specifically indicates that BCG strain differences can modulate the infant response to subsequent, unrelated exposures to antigens, including vaccines (and presumably, pathogens).
There was striking disparity in BCG scar frequency between groups, with an almost two-fold increase in scarring frequency in the BCG-Denmark group compared to the BCG-Russia group. The overall proportion with scars was 59%, despite 100% immunisation coverage at birth. This is much lower than observed scar prevalence in other populations [25,27,28,40]
and is unlikely to be attributable to poor immunisation technique considering that 93% of the BCG-Denmark group developed a scar (p
< 0.001). This analysis may be evidence that the association between BCG scar frequency and immunisation status is strain-dependent. BCG scars have often been used in research to identify BCG immunised individuals, which may be a valid method in a population uniformly immunised with one strain, such as BCG-Denmark, which causes the majority of vaccinees to scar. However, in populations immunised with a strain that causes fewer scars, scarring may reflect an individual's immune response to the vaccine rather than immunisation status, leading to many misclassifications. In countries using multiple strains, identifying individuals by scar status may give results reflecting the effects of one strain and not the whole immunised population. Although correlations between scar size and cytokine responses have been demonstrated at 4 years of age 
, it is unsurprising that no relationship was shown here, as BCG scars are still very small at one year.
Studies in Guinea Bissau have demonstrated an association between scar development after BCG immunisation and benefiting from its non-specific effects [14,25–27]
. However, our results show no correlation between scarring and non-specific cytokine responses, with only higher mycobacteria-specific IFN-γ and IL-13 responses differentiating those with a scar from those without. BCG strain did influence both non-specific immune responses and scar development, suggesting that BCG strain could be a confounder in the relationship between scarring and non-specific responses. For example, the BCG-Denmark strain caused both higher IFN-γ responses to non-specific stimuli and also a greater frequency of scarring. The infants’ sex modified the effect of BCG strain on responses to tetanus toxoid, but not to either mycobacteria-specific antigen. This finding is in keeping with reports that girls may experience more non-specific BCG effects than boys [14,26,35,36]
although a mechanism for this phenomenon has not been established 
This study was underpowered to detect differences in mortality. However, significant differences were detected between the proportions of each group that experienced an adverse event, the highest of which occurred in the BCG-Denmark group. As BCG-Denmark stimulated the highest cytokine responses, it is possible that there may be a trade-off between immunogenicity and adverse event induction, although the small number of events warrants caution in interpreting this relationship.
Our results emphasise the importance of identifying and adjusting for the strain of BCG used in studies of vaccine efficacy, or of correlates of protection, whenever BCG is employed as part of a vaccination strategy. This includes studies evaluating novel vaccines that employ a prime–boost strategy, as the choice of priming BCG strain may influence the results. Our data also suggests that BCG strain may influence outcomes when employing BCG as a vector for vaccines against other pathogens. Importantly, the choice of BCG strain may have clinical effects beyond the protection against TB. Further large-scale comparative investigation of BCG strains with clinical primary outcomes would be valuable.
This analysis was not part of our original trial design, so infants were not randomised to receive different BCG strains. This may have led to potential confounders, for example, due to different seasonal exposures to infections, which we could not account for. However, we did identify differences in maternal helminth and infant malaria status between the groups and we adjusted for these variables in the analysis; adjusted results were similar to crude findings. One-year olds were appropriate subjects as it has been shown that IFN-γ, IL-5, IL-13 and IL-10 responses to BCG given at birth are detectable at one year with some effects waning by two years 
. However, it was not possible to analyse TB outcomes or long-term effects. Further work will include a repeated analysis of the same cohort at five years, assessing TB prevalence and incidence as well as non-TB illnesses and overall mortality. This may provide the warranted longitudinal evidence of whether or not strain-dependent effects observed at the molecular level translate to clinical outcomes in this cohort. In the meantime, whenever multiple BCG strains are used in future research, or when the effects of BCG or other immunisation regimes are compared in different populations, accounting for BCG strain is vital.