Optimisation, harmonisation and standardisation of the direct mycobacterial growth inhibition assay using cryopreserved human peripheral blood mononuclear cells.

A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development

Cell-Mediated Immune Responses to in vivo -Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups

A quarter of the global human population is estimated to be latently infected by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). TB remains the global leading cause of death by a single pathogen and ranks among the top-10 causes of overall global mortality. Current immunodiagnostic tests cannot discriminate between latent, active and past TB, nor predict progression of latent infection to active disease. The only registered TB vaccine, Bacillus Calmette-Guérin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-γ centered approaches, have been proposed as targets for new TB-diagnostic tests or -vaccines. Recently, however, we identified novel Mtb antigens capable of eliciting multiple cytokines, including antigens that did not induce IFN-γ but several other cytokines. These antigens had been selected based on high Mtb gene-expression in the lung in vivo, and have been termed in vivo expressed (IVE-TB) antigens. Here, we extend and validate our previous findings in an independent Southern European cohort, consisting of adults and adolescents with either LTBI or TB. Our results confirm that responses to IVE-TB antigens, and also DosR-regulon and Rpf stage-specific Mtb antigens are marked by multiple cytokines, including strong responses, such as for TNF-α, in the absence of detectable IFN-γ production. Except for TNF-α, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-α+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-α+ cells clusters identified, those with CD8+TEMRA or CD8+CD4+ phenotypes, defined by the expression of multiple intracellular markers, were the most prominent in adult LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb

Identification of circulating monocytes as producers of tuberculosis disease biomarker C1q

Tuberculosis (TB) is a prevalent disease causing an estimated 1.6 million deaths and 10.6 million new cases annually. Discriminating TB disease from differential diagnoses can be complex, particularly in the field. Increased levels of complement component C1q in serum have been identified as a specific and accessible biomarker for TB disease but the source of C1q in circulation has not been identified. Here, data and samples previously collected from human cohorts, a clinical trial and a non-human primate study were used to identify cells producing C1q in circulation. Cell subset frequencies were correlated with serum C1q levels and combined with single cell RNA sequencing and flow cytometry analyses. This identified monocytes as C1q producers in circulation, with a pronounced expression of C1q in classical and intermediate monocytes and variable expression in non-classical monocytes

The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts

Mycobacterium tuberculosis peptides presented by HLA-E molecules are targets for human CD8 T-cells with cytotoxic as well as regulatory activity

Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8(+) T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8(+) T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8(+) T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-beta. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8(+) T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity

TBVAC2020: Advancing tuberculosis vaccines from discovery to clinical development

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal

INNOVATIVE STRATEGIES TO IDENTIFY M. TUBERCULOSIS ANTIGENS AND EPITOPES USING GENOME-WIDE ANALYSES

In view of the fact that only a small part of the Mtb expressome has been explored for identification of antigens capable of activating human T-cell responses, which is critically required for the design of better TB vaccination strategies, more emphasis should be placed on innovative ways to discover new Mtb antigens and explore their function at the several stages of infection. Better protective antigens for TB vaccines are urgently needed, also in view of the disappointing results of the MVA85 vaccine which failed to induce additional protection in BCG vaccinated infants [54]. Moreover, immune responses to relevant antigens may be useful to identify TB-specific biomarker signatures. Here we describe the potency of novel tools and strategies to reveal such Mtb antigens. Using proteins specific for different Mtb infection phases, many new antigens of the latency-associated Mtb DosR regulon as well as Rpf proteins, associated with resuscitating TB, were discovered that were recognized by CD4+ and CD8+ T-cells. Furthermore, by employing MHC binding algorithms and bioinformatics combined with high throughput human T-cell screens and tetramers, HLA-class Ia restricted poly-functional CD8+ T-cells were identified in TB patients. Comparable methods, led to the identification of HLA-E-restricted Mtb epitopes recognized by CD8+ T-cells. A genome-wide unbiased antigen discovery approach was applied to analyse the in vivo Mtb gene expression profiles in the lungs of mice, resulting in the identification of IVE-TB antigens, which are expressed during infection in the lung, the main target organ of Mtb. IVE-TB antigens induce strong T cell responses in long-term latently Mtb infected individuals, and represent an interesting new group of TB antigens for vaccination. In summary, new tools have helped expand our view on the Mtb antigenome involved in human cellular immunity and provided new candidates for TB vaccination. <br/

Treg-cell marker frequency and density are increased on live BCG-activated CD8⁺ vs. CD4⁺ T cells.

<p>A: BCG induces Treg-cell marker expression on CD4⁺ and CD8⁺ T cells; after live BCG stimulation the percentage of total CD8⁺ T cells expressing CD25, Foxp3, CD39, LAG-3 or CCL4 is significantly higher compared to CD4⁺ T cells, depicted here as frequency of CD8⁺ or CD4⁺ population. Differences in Treg marker expression between heatkilled BCG–activated CD8⁺ vs. CD4⁺ T cells were not significant, except for expression of CCL4; CCL4 expression was also significantly higher on CD8⁺ T cells compared to CD4⁺ T cells in samples not stimulated with BCG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192.s001" target="_blank">Fig. S1</a>) (<i>*p</i> < 0.05, Wilcoxon signed-ranks test). B: Mean fluorescence intensities (MFIs) of CD25 and CD39 are increased on live BCG-activated CD8⁺ T cells as compared to CD4⁺ T cells. Gating was performed as demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone-0094192-g001" target="_blank">figure 1A</a>. To assess differences in intrinsic intensity of expression on CD4⁺ and CD8⁺ T cells, respectively, MFIs of positive Treg marker populations in samples not stimulated with BCG were compared; this was similar on CD4⁺ and CD8⁺ T cells for MFIs of CD25, Foxp3 and CD39. Data are representative of seven <i>in vitro</i> PPD-responders six days after heatkilled or live BCG stimulation (*<i>p</i> < 0.05; Wilcoxon signed-ranks test).</p

Heatkilled vs. live BCG-activated expression of Treg-cell markers on CD4⁺ and CD8⁺ T cells.

<p>A: Gating strategy: cells were gated on single cells, live lymphocytes, CD3⁺ and CD4⁺CD8⁻ vs. CD4⁻CD8⁺. Demonstrated is the synchronized gating on the positive population of interest for CD4⁺CD8⁻ and CD8⁺CD4⁻ T cells; here the CD25-positive population. B: Heatkilled and live BCG activate CD25⁺Foxp3⁺ and LAG-3⁺CD39⁺ T cells. Expression of regulatory T cell markers on CD4⁺ and CD8⁺ T cells of <i>in vitro</i> PPD responders was analysed by flowcytometry six days after heatkilled or live BCG stimulation. For each donor gating was compared to samples not stimulated with BCG (demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192.s001" target="_blank">Fig. S1</a>). Data are representative of seven responders.</p