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

Abstract

Objective: This study determined the effects of chemical adjuvants, incomplete Freund's adjuvant (IFA) and aluminum hydroxide (Alum), mycobacteria, and a DNA plasmid as delivery systems on the induction of protective Th1 (interferon-gamma (IFN-γ)) and nonprotective Th2 (IL-5) and Treg (IL-10) cytokine responses to Rv3619c and its peptides. Rv3619c is an immunodominant Mycobacterium tuberculosis-specific antigen and belongs to the early-secreted antigenic target of 6 kDa-family of proteins. Delivery systems are needed to deliver such antigens in animal models and induce protective immune responses.

Methods: 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 mice immunized with rv3619c using different delivery systems were stimulated in vitro with synthetic peptides (P1 to P6) of Rv3619c, and secreted cytokines were estimated by ELISA.

Results: The recombinant M. smegmatis and DNA plasmid induced the secretion of the protective cytokine IFN-γ in response to peptide-pool of Rv3619c and all the individual peptides, whereas rv3619c/IFA induced the secretion of IFN-γ in response to the peptide pool, and the peptides P5 and P6. However, the secretions of the nonprotective cytokines IL-5 and IL-10 were induced to none of the peptides with the delivery systems used.

Conclusion: Rv3619c is a major antigen of M. tuberculosis with multiple immunogenic epitopes; however, immune responses to individual epitopes can vary based on delivery systems used.

Keywords: Delivery systems; IFN-γ; Mycobacterium tuberculosis; Peptides; Rv3619c.

Fig. 1

aSDS-analysis of purified Rv3619c protein (9.8 kDa). Lane M: Prestained low molecular weight marker. Lanes 1–3: GST-free Rv3619c proteins. bAgarose gel electrophoresis of PCR-amplified products using genomic DNA isolated from rM. smegmatis transformed with pDE22/rv3619c, DNase-treated genomic DNA, and cDNA (reverse-transcriptase-PCR). Lane M: 100 bp marker. Lane 1: Genomic DNA amplified with Rv3619c primers (285 bp product). Lane 2: Genomic DNA treated with DNase and amplified with rv3619c primers (no product) Lane 3: cDNA amplified with rv3619c primers (285 bp product).

Fig. 2

Cytokine concentrations (pg/mL) in supernatants of cultured spleen cells from mice administered with PBS, Rv3619c/IFA, Rv3619c/Alum, wild-type M. smegmatis, rM. smegmatis/rv3619c, and rDNA plasmid/rv3619c and stimulated with the peptide pool of Rv3619c. Spleen cells obtained from immunized mice were cultured in triplicates in the absence of any stimulant (control) and in the presence of stimulants (experimental), i.e., pool of Rv3619c peptides (P1 to P6 [PP]) covering the sequences of Rv3619c protein. The supernatants were collected on day 6. The culture supernatants were tested for secreted IFN-γ, IL-5, and IL-10 in duplicate wells of 96-well plates by ELISA. The concentration of cytokines in response to pool of Rv3619c peptides were considered significant (*) with quantities >100 pg/mL and the ratio of experimental/control >2 [9, 11]. E/C, experimental/control.

Fig. 3

Cytokine concentrations (pg/mL) in supernatant of cultured spleen cells from mice immunized with Rv3619c/IFA (a), Rv3619c/Alum (b), rM. smegmatis/rv3619c (c), and rDNA plasmid/rv3619c (d) and stimulated with the individual peptides (P1 to P6) of Rv3619c. Spleen cells obtained from immunized mice were cultured in triplicates in the absence of any stimulant (control) and in the presence of stimulants (experimental), i.e., individual peptides of Rv3619c (P1 to P6) covering the sequences of Rv3619c protein. The supernatants were collected on day 6. The culture supernatants were tested for secreted IFN-γ, IL-5, and IL-10 in duplicate wells of 96-well plates by ELISA. The concentration of cytokines in response to pool of Rv3619c peptides were considered significant (*) with quantities >100 pg/mL and the ratio of experimental/control >2 [9, 11]. E/C, experimental/control.