Age-Related Immunoreactivity Profiles to Diverse Mycobacterial Antigens in BCG-Vaccinated Chinese Population

Abstract

Long-term immunoreactivity to mycobacterial antigens in Bovis Calmette-Guérin (BCG)-vaccinated population is not well investigated. Herein, 361 volunteer healthy donors (HDs) with neonatal BCG vaccination from Shanghai region (China) were enrolled. They were subdivided into ESAT-6/CFP10^- (E6C10^-) and ESAT-6/CFP10⁺ (E6C10⁺) groups based on gamma-interferon release assays (IGRAs). Three mycobacterial antigens, including Rv0934, Rv3006, and Rv3841, were subjected to the determination of immunoreactivity by ELISPOT assay. The immunoreactivities to three mycobacterial antigens were firstly compared among TB patients (N=39), E6C10⁺ HDs (N=78, 21.61% of HDs) and E6C10^- HDs (N=283, 78.39% of HDs). It was revealed that Rv3006 was dominant upon M.tb infection, while Rv3841 was likely to be more responsive upon latent TB infection. In E6C10^- population, the immunoreactivity to Rv3841 maintained along with aging, whereas those to Rv3006 and Rv0934 attenuated in E6C10^- HDs older than 45 years old. Our study implies the shift of dominant antigens at different infection statuses, providing the clues for the selection of mycobacterial antigens in vaccine development and precision revaccination in the future.

Keywords: BCG vaccination; aging; healthy population; immunoreactive profiles; mycobacterial proteins.

Figure 1

Expression and identification of recombinant proteins Rv0934, Rv3006, and Rv3841. (A) Identification of the purity of prokaryotically expressed Rv0934, Rv3006, and Rv3841 recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie brilliant blue. (B) Verification of purified Rv0934, Rv3006, and Rv3841 proteins by western blotting. Purified His-tagged proteins were confirmed with mouse anti-6×His tag antibody and HRP conjugated goat anti-mouse IgG antibody. Molecular mass: Rv0934 Protein: 38 kDa; Rv3006 Protein:38 kDa; Rv3841 Protein: 20 kDa.

Figure 2

Different gamma-interferon (IFN-γ) response patterns targeting Rv0934, Rv3006 and Rv3841. (A) Representative results of enzyme-linked immunospot (ELISPOT) assay. Peripheral blood mononuclear cells (PBMCs) were stimulated with different antigens for 20 h, including M.tb-specific ESAT-6 peptide (E6p) and CFP10 peptide (C10p) as well as three mycobacterial recombinant proteins. Culture medium served as the negative control (BL), while phytohemagglutinin (PHA) as the positive control. (B–D) Comparison of IFN-γ responses of Rv0934, Rv3006, and Rv3841 in E6C10^- population (B), E6C10⁺ population (C), and in tuberculosis (TB) patients (D). The p-values were calculated using the Kruskal-Wallis test and Dunn’s multiple comparison test. The p-values above the lines are the p-values of the Kruskal-Wallis test. The p-values above capped lines are the adjusted p-value of Dunn’s multiple comparison test. ****p < 0.0001.

Figure 3

Numbers of mycobacterial antigen-specific gamma-interferon (IFN-γ) releasing cells in E6C10^- population. (A–C) Rv0934 (A)-, Rv3006 (B)-, and Rv3841 (C)- specific IFN-γ releasing cell numbers in peripheral blood mononuclear cells (PBMCs) from different age groups of E6C10^- subjects. The p-values were calculated using the Kruskal-Wallis test and Dunn’s multiple comparison test. The p-values above the lines are the p-values of the Kruskal-Wallis test. The p-values above capped lines are the adjusted p-value of Dunn’s multiple comparison test. ****p < 0.0001. ***p < 0.001, **p < 0.01. *p < 0.05.

Figure 4

Numbers of mycobacterial antigen-specific gamma-interferon (IFN-γ) releasing cells in E6C10⁺ population. (A–C) Rv0934 (A)-, Rv3006 (B)-, and Rv3841 (C)- specific IFN-γ releasing cell numbers in peripheral blood mononuclear cells (PBMCs) from different age groups of E6C10⁺ subjects. The p-values were calculated using the Kruskal-Wallis test and Dunn’s multiple comparison test.

Figure 5

Numbers of mycobacterial antigen-specific gamma-interferon (IFN-γ) releasing cells in TB patients. (A–C) Rv0934 (A)-, Rv3006 (B)-, and Rv3841 (C)- specific IFN-γ releasing cell numbers in peripheral blood mononuclear cells (PBMCs) from different age groups of tuberculosis (TB) patients. The p-values were calculated using the Kruskal-Wallis test.