Supplementary Material for: The Effect of Delivery Systems on the Induction of T Helper 1 Cell Response to an ESAT6-Like Protein Rv3619c and Identification of its Immunodominant Peptides

Rv3619c is an immunodominant Mycobacterium tuberculosis-specific antigen and belongs to the ESAT6-family of proteins. Delivery systems are needed to deliver such strong antigen in animal model and induce protective immune responses. Hypothesis. This study determined the effect of chemical adjuvants (IFA and Alum), mycobacteria, and a DNA plasmid as delivery systems on the induction of protective Th1 (IFN-ɣ), and pathologic Th2 (IL-5) and Treg (IL-10) cytokine responses to Rv3619c and its peptides. The Rv3619c gene was amplified from the genomic DNA of M. tuberculosis and cloned into appropriate vectors for expression in Escherichia coli, Mycobacterium smegmatis, and eukaryotic cells. Spleen cells from the mice immunized with Rv3619c using different delivery systems were stimulated in vitro with synthetic peptides (P1 to P6) of Rv3619c, and secretions of cytokines were estimated by ELISA. The results showed that the recombinant M. smegmatis and DNA plasmid induced the secretion of protective cytokine IFN-ɣ in response to peptide-pool of Rv3619c and all the individual peptides, whereas Rv3619c/IFA induced the secretion of IFN-ɣ in response to the peptide pool, and the peptides P5 and P6. However, the secretions of pathologic cytokines IL-5 and IL-10 were induced to none of the peptides with the delivery systems used. These results show that Rv3619c is a major antigen of M. tuberculosis with multiple immunogenic epitopes but the immune responses to individual epitopes can vary based on delivery systems used.