Identification and selection of novel mycobacterial antigens using immunopeptidomics and immunoinformatics for the development of subunit vaccines against tuberculosis
Keywords: tuberculosis
immunopeptidomics
antigen discovery

In response to the urgent need for an effective vaccine against tuberculosis (TB), mass spectrometry-based immunopeptidomics was employed to identify novel mycobacterial antigens. Human macrophages (differentiated THP-1 cells) were utilised as an in-vitro model of TB, in which mycobacterial peptides presented on major histocompatibility complex (MHC) molecules were identified. As a result, 64 and 81 mycobacterial proteins derived from the Bacille Calmette-Guérin (BCG) vaccine and Mycobacterium tuberculosis (MTB) were discovered, respectively. The identified proteins were prioritised based on their T-cell epitope repertoire through the use of immunoinformatics tools in silico, resulting in a list of potential candidate antigens. This approach efficiently facilitated the identification and prioritisation of novel mycobacterial antigens, in which T-cell responses and the diversity of human leukocyte antigen (HLA) alleles were considered. The top candidate antigens (hisD, metE and mmpL12) were selected for screening in mice through the application of mycobacterial growth inhibitory assays (MGIAs) to evaluate their protective properties. MGIA was used as an ex-vivo surrogate assay for an in-vivo MTB challenge experiment. The mouse groups that were vaccinated with metE (either in the form of plasmid DNA or as a protein with adjuvant) conferred protection compared with unvaccinated mice. The metE-DNA vaccine induced polyfunctional CD4+ T cells expressing interferon-gamma (IFNγ), tumour necrosis factor-alpha (TNFα), and interleukin-2 (IL-2), whereas the metE-AddaS03 vaccine elicited a range of CD4+ T cells that expressed TNFα, IL-2, IL-4, and IL-17. Both the metE-DNA and metE-AddaS03 vaccines significantly induced the production of immunoglobulin G (IgG) antibodies compared with the naïve group; when the vaccines were compared, the antibody concentration that was induced by the metE-AddaS03 vaccine was 16-fold higher than that induced by the metE-DNA vaccine. The findings of this work show that the metE protein is a novel protective candidate that merits further development for inclusion in a subunit vaccine against TB.
Interestingly, IFNγ was found to enhance the expression and the total number of identified peptides that were associated with HLA-DR significantly compared with HLA-DQ and HLA-DP. The effects of mycobacterial infection and IFNγ treatment on the surface expression of HLA-DR alleles (HLA-DRB1*0101, -DRB1*1501 and - DRB5*0101) expressed by THP-1 cells were investigated quantitatively. Mass spectrometry-based quantitative proteomics was used to analyse the expression levels of HLA-DR proteins obtained from the same samples used for immunopeptidomics. Without IFNγ treatment, DRB1*0101 was dominantly expressed compared with DRB1*1501 and DRB5*0101. In contrast, when the sample was treated with IFNγ, DRB1*1501 was up-regulated more than DRB1*0101. Compared with uninfected samples, the levels of HLA-DRA1 and -DRB1*0101 showed a decreasing trend upon infection, whereas levels of DRB1*1501 and DRB5*0101 increased or remained unchanged. When the mycobacterial peptides were predicted according to their binding affinity, 55% of peptides were assigned as ligands for DRB1*0101, compared with about 24.3% and 20.7% assigned as ligands for DRB1*1501 and DRB5*0101, respectively. To expand this further to the whole sequence, 76% of the tested antigens (47 antigens) were predicted to contain more peptides for DRB1*0101 than for DRB1*1501. The data collected in this work suggest that mycobacteria may evade Tcell recognition as they target HLA-DR alleles that are more likely to present their antigens (i.e. they inhibit the HLA-DRB1*0101 allele and avoid being presented by the DRB1*1501 allele).