Identification of antigens presented by MHC for vaccines against tuberculosis

Abstract

Mycobacterium tuberculosis (M.tb) is responsible for more deaths globally than any other pathogen. The only available vaccine, bacillus Calmette-Guérin (BCG), has variable efficacy throughout the world. A more effective vaccine is urgently needed. The immune response against tuberculosis relies, at least in part, on CD4⁺ T cells. Protective vaccines require the induction of antigen-specific CD4⁺ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. In order to identify mycobacterial antigens bound to MHC, we have immunoprecipitated MHC class-I and class-II complexes from THP-1 macrophages infected with BCG, purified MHC class-I and MHC class-II peptides and analysed them by liquid chromatography tandem mass spectrometry. We have successfully identified 94 mycobacterial peptides presented by MHC-II and 43 presented by MHC-I, from 76 and 41 antigens, respectively. These antigens were found to be highly expressed in infected macrophages. Gene ontology analysis suggests most of these antigens are associated with membranes and involved in lipid biosynthesis and transport. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cell from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying new candidate antigens.

Keywords: Tuberculosis; Vaccines.

Fig. 1. Peptide identification and selection of vaccine candidates.

a Distribution of unique human and BCG peptides identified in each experiment. Distribution of unique human peptides from the four experiments combined, identified in function of length (b), and according to the netMHCpan rank (c). The best match HLA binding allele for each peptide was determined and selected. MHC-I peptides with a rank ≤ 0.5 or between 0.5 and 3.0 were considered strong or weak binders, respectively. MHC-II peptides with a rank ≤ 2 or between 2 and 10 were considered strong or weak binders, respectively. The remaining peptides were excluded. The alleles identified as best binders were HLA-A*02:01, HLA-B*15:11, HLA-B*15:15, HLA-C*03:03, HLA-C*03:13, HLA-DRB1*01:01, HLA-DRB1*15:01 and HLA-DRB5*01:01. d Selection of unique BCG peptides from total peptides following the pipeline for identification. A Peaks score cut-off of >15 was used for the peptide identification. After applying our data analysis pipeline, peptides were selected for presence in more than 1 sample (I, II). These were further selected if found in different experiments (III, IV) or if found in more than one sample but not in different experiments (V, VI). If peptides were not present in more than one sample, they were further down-selected into different peptide sequences found on the same antigen (VII, VIII) or found only once (IX, X). MHC-II peptides also presented nested sequences included in II and IV. e Total BCG antigens identified by MHC-I and MHC-II. f Table showing selected antigens for vaccine production.

Fig. 2. Gene expression and gene ontology of antigens identified.

Associated genes of MHC-I (a) and MHC-II presented peptides (c) are highly expressed in M. bovis BCG RNA extracted after 24 h infection of THP-1 cells. Normalised expression levels of M. bovis BCG genes were measured by RNA-seq. Genes encoding peptides detected on MHC-I (b) or MHC-II (d) are significantly higher expressed compared to all genes in M. bovis BCG RNA extracted after 24 h infection of THP-1 cells. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles) and the centre line corresponds to the median. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Gene ontology of identified antigens. Proteins of MHC-I and MHC-II associated peptides (Table 2) were linked to EBI QuickGO Gene ontology clusters and displayed as chord plots for the three aspects Biological Process (e, h), Molecular Function (f, i) and Cellular Component (g, j). Proteins without a GO category were labelled as Unknown. To prevent overplotting, only the first 22 GO categories are shown and all lower categories as determined by enrichment p-values were labelled as “Other”. P-values were estimated using a Mann–Whitney test.

Fig. 3. Peptide-specific responses of IFNγ secreting PBMCs from patient groups.

ELISpots of PBMCs from 14 BCG-vaccinated individuals HLA-matched (n = 5), BCG-vaccinated individuals non-HLA matched (n = 8), latently infected individuals HLA-matched (n = 7), latently infected individuals non-HLA matched (n = 10 in (a, b, c, d, e); n = 9 in (f) and tuberculosis active patients (n = 5 in (a, b, c, d, e); n = 4 in (f) were used. Cells were stimulated with 2μg/ml of individual or pooled peptides or 20μg/ml PPD, or left unstimulated, with 3 × 10⁵ cells per well in duplicates. Cells were stimulated with (a) fbpA_44–51 FSRPGLPV, (b) BCG_3870c_4–12 LAASLLSRV, (c) fas_57–65 GIETELATL, (d) iniC_191–200 QIGGLVGGSV, (e) groL2_61–75 LEDPYEKIGAELVKE and (f) a pool of peptides containing these five sequences and mmpL12_396–403 AGCTLLIR and atpA_333–340 KANDISAY (a peptide identified with a Peaks score of 14.54 hence excluded from the analysis). Each dot represents the results from each peptide. The error bars show the mean and standard errors of the mean. Statistical analysis performed with Two-tailed Mann–Whitney U test, and statistically significant differences between groups are represented as *p < 0.05, **p < 0.01.

Fig. 4. Protective efficacy of selected antigens alone or in combination, against a challenge with an aerosol of M.tb Erdman.

a Experimental design. All groups of CB6F1 mice were vaccinated with BCG, intradermally. Ten weeks later, mice were boosted with intranasal ChAdOx1, followed by intradermal MVA, 4 weeks later, the BCM schedule. A group of mice was vaccinated with BCG only and another group left unvaccinated. Four weeks after BCM, all mice were challenged with an aerosol of M.tb Erdman (100 CFU per animal). Four weeks later, animals were culled, lung and spleen harvested and CFU counted. CFU from lung (b) and spleen (c), from the first experiment comprising of BCM expressing single antigens (glfT2, fas, iniB or Ag85A). CFU from lung (d) and spleen (e), from the second experiment consisting of BCM expressing single antigens (glfT2, fas, or iniB) or a combination of these three antigens (GIF) or a combination of GIF plus Ag85A and PPE15 (GIFPA). CFU, colony forming units, n = 8. Each symbol represents one animal, the error bars represent the median and the columns represent the interquartile range. Kruskal–Wallis followed by Dunn’s multiple comparison test (black bars) as well as Mann–Whitney U test (grey bars) were used to assess significance, *p < 0.05, **p < 0.01.