Whole blood RNA signatures in tuberculosis patients receiving H56:IC31 vaccine as adjunctive therapy

The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR using 96.96 IFC chips on the Biomark HD system (Standard BioTools), as described by the manufacturer. Data was analyzed using Standard BioTools Real-Time PCR Analysis Software (version 4.1.3). A Ct value < 35 was determined as the cut-off for reliable detection. Relative target gene expression was determined by calculating ∆Ct using GAPDH as a reference gene. GAPDH-normalized gene expression data were corrected for batch effects by using the ComBat function implemented in the R sva package 3.48.0 using the default parametric adjustment mode. 

Longitudinal Differential Expression Analysis (DEA) was performed comparing i) H56:IC31 group versus Controls; and ii) Responders versus Controls, at all post-vaccine time points (from day 84 until day 238). The non-parametric Mann-Whitney U-test with Benjamini-Hochberg correction for multiple testing was applied in all comparisons. A p-value <0.05 was set as the threshold for the identification of Differential Expression Genes (DEGs).