INVESTIGATING THE ROLE OF INNATE IMMUNITY IN MEDIATING THE NON-SPECIFIC EFFECTS OF BACILLE CALMETTE-GUÉRIN VACCINE

Abstract

Background: Epidemiological evidence suggests that Bacillus Calmette-Guerin (BCG) vaccine exerts non-specific (heterologous) effects in infants; decreasing neonatal mortality in high-mortality settings and preventing allergy and morbidity from infection in developed countries. New tuberculosis (TB) vaccines could potentially lack these beneficial effects. Immune mechanisms underlying the non-specific effects of BCG vaccine have been linked to ‘trained immunity’ or innate immune memory.

Aims: Part 1: To investigate whether neonatal BCG vaccination alters the immune response to heterologous pathogens and Toll-like receptor (TLR) ligands in (i) the neonatal period and (ii) infancy. Part 2: To use a systems vaccinology approach to identify innate immune signatures underlying the non-specific effects of BCG vaccine.

Methods: Part 1: In the Melbourne Infant Study: BCG for allergy and infection reduction (MIS BAIR), 1272 infants were randomised to receive BCG vaccine or no BCG vaccine within the first 10 days of life. A subset of participants was recruited to an immunological sub-study. A whole blood stimulation assay of ‘specific’ mycobacterial antigens, heterologous bacterial or fungal antigens and TLR ligands was used to interrogate cytokine responses at 7 days (n=212) and 7 months (n=167) post randomisation. Part 2: In the BabyBAIR study, 44 infants who were BCG vaccinated prior to travel to a TB-endemic area were recruited. Blood was collected from participants prior to BCG vaccination, and at 7 days and 3 months post vaccination. Cytokine responses and cell populations were analysed following in vitro stimulation of whole blood as above. RNASeq was also done on whole blood and the transcriptome was analysed for differentially expressed genes. Pathway analysis was done using functional gene set enrichment analysis (fGSEA) at each time point compared to baseline.

Results: In the MIS BAIR study, infants who were BCG-vaccinated had significant differences in their heterologous cytokine responses at both 7 days and 7 months post randomisation compared to BCG-naive infants. At 7 days post randomisation, compared to BCG-naive neonates, BCG-vaccinated neonates had evidence of a pro-inflammatory bias in RPMI-stimulated (nil) samples. Following heterologous stimulation, BCG-vaccinated neonates had decreased chemokine (MCP-1, MIP-1alpha, MIP-1beta, IL-1RA, IL-6 and IL-10 responses following stimulation of TLR2 (PEPG) and TLR7/8 (R848). At 7 months post randomisation, compared to BCG-naive infants, BCG-vaccinated infants, had decreased IFN-gamma responses to stimulation with heterologous pathogens. Decreased IFN-γ responses in the BCG-vaccinated group were attributable to a reduction in the proportion of individuals mounting an IFN-gamma response. Heterologous cytokine responses were modified by sex and maternal BCG vaccination status in both neonates and older infants.

In the BabyBAIR study, longitudinal cytokine analysis showed BCG vaccination to be associated with a pro-inflammatory bias at baseline (prior to BCG vaccination), a robust pro-inflammatory heterologous response at 7 days post BCG vaccination and downregulation of pro-inflammatory cytokines 3 months post BCG vaccination compared to baseline. Flow cytometry suggested that both myeloid and monocyte derived dendritic cells were associated with the observed heterologous cytokine responses. RNASeq analysis of the whole blood transcriptome following BCG vaccination indicated widespread changes in innate immune signalling pathways and identified several potential mechanisms by which BCG vaccine could mediate its beneficial heterologous effects.

Conclusions: Neonatal BCG vaccination leads to significant changes in the immune phenotype of vaccinated individuals. Following in vitro stimulation with heterologous pathogens, BCG-vaccinated infants have altered immune responses which might improve regulation of the inflammatory response during acute infection and a subsequent reduction in all-cause mortality. These results support the paradigm of BCG-induced trained immunity and provide additional information regarding the nature of the response in neonates and between different classes of pathogens.