Nina Marie Birk Eriksen 2017

Eriksen, Nina Marie Birk. 2017. Immune modulating effects of BCG vaccination at birth on thymic size and subsets of T and B lymphocytes.

Mounting evidence suggests that the definition of a vaccine as ` a biological preparation that improves immunity to a particular disease´ is too narrow - and that the view of vaccines as one-target immune modulators, may have to be reconsidered. Observational studies6–9 and recent randomized controlled trials10,11, suggest that the vaccine Bacillus Calmette-Guérin (BCG) has beneficial effects beyond the specific protection against tuberculosis (TB); protecting against both morbidity and mortality in low income countries. These effects are termed ‘non-specific effects’ (NSEs). The immunological mechanisms behind these observed NSEs are not fully understood. Since BCG is not part of the routine vaccination program in Denmark, assessing NSEs in a randomized trial was ethically justified. This lead to the conduction of The Danish Calmette Study; a randomized clinical multicenter trial, investigating the effects of BCG or no BCG at birth on clinical and immunological outcomes in a high income setting13. Between October 2012 and November 2015, 4262 infants were randomized 1:1 to BCG or no intervention and followed clinically at 3 and 13 months of age. The focus of the present thesis was the immune modulating effects of BCG on the adaptive immune response in a subgroup of infants. The aims were a) to investigate if BCG would increase thymic size at the age of 3 months, and further to assess the associations between thymic size and thymic output and also between thymic size and number of infections within the first 3 months of life; b) to investigate the effects of BCG on a broad panel of T and B cell subsets assessed by flow cytometry 4 (±)2 days after randomization, and at 3 and 13 months; c) to investigate the association between the post-BCG vaccination skin reaction (wheal, bulge, no reaction) and the probability of developing a scar, furthermore the association between wheal and scar size and last the association between wheal size and the probability of developing a scar. 

In 301 infants thymic size was measured as the thymic index (TI) and thymic weight index (TWI) by transsternal ultrasound at birth and at 3 months of age. Our hypothesis of a BCG induced increase in thymic size was rejected, as no effect was found of BCG on neither TI (GMR: 0.92, 95% CI (0.84–0.99)) nor TWI (GMR: 0.91, 95% CI (0.85–0.99)). However, post hoc analysis in infants having both TI and TWI as well as T and B cells assessed (n=112), showed thymic size positively associated with thymic output (CD4+, CD4+ RTEs, and lymphocytes) at birth. No associations between cell subsets and TI or TWI were found at 3 months of age. Post-hoc analysis showed TI and TWI at birth negatively associated with numbers of 67 subsequent infections at 3 months of age. In the evaluation of T and B cell subsets in 118 infants, no effect was found on the primary outcome: cell subsets measured 4 (±)2 days after randomization. No overall effect was seen in cells characterized as naïve, and concerning thymic output or regulatory cells either. BCG was however positively associated with secondary outcomes of effector memory T cells (CD4+CD45RA-CD27+CCR7-) at 3 months of age, and negatively associated with apoptotic T cells (CD4+CD95+CD28-) and late differentiated T cells (HLADR+CD38+) at 13 months of age. Potential effect modification by time of vaccination (within days after birth) and sex of the child was detected in cells of T regulatory cells (CD19+CD24highCD38high B cells and T regulatory cells). Our primary hypothesis was rejected; however our results suggest that BCG may induce T cell memory at 3 months of age. This corresponds to the induction of BCG specific memory T cells as described in studies concerning the specific effects of BCG in TB protection22,32. Finally post-BCG vaccination skin reaction categories (wheal, bulge, or no reaction) predicted significantly different probabilities of developing a scar (96%, 87%, and 56%). Moreover, an increase in wheal size increased both scar size and the chance of getting a scar. In conclusion, BCG did not affect the primary outcomes of thymic size or T and B subsets at 4 days post randomization. Overall limited impact was found of neonatal BCG vaccination on lymphocyte subsets in healthy infants within the first 13 months of life in a high-income setting. This corresponds to the findings of limited effect of BCG on the clinical outcomes and other immunological outcomes within the Danish Calmette Study. We demonstrated that the skin reaction at the injection site is highly important for the probability of developing a scar after neonatal BCG in a high-income setting. Training in correct injection technique may improve the probability of getting a BCG scar and registration of the BCG skin reaction category immediately following vaccination may be used to identify infants at risk of scar failure.