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Introduction: Sarcoidosis is considered an idiopathic granulomatous disease, although similar immunological and clinical features with tuberculosis (TB) suggest mycobacterial involvement in its pathogenesis. High-content peptide microarrays (HCPM) may help to decipher mycobacteria-specific antibody reactivity in sarcoidosis., Methods: Serum samples from patients with sarcoidosis, Löfgren's syndrome, and TB, as well as from healthy individuals (12/group), were tested on HCPM containing 5964 individual peptides spanning 154 Mycobacterium tuberculosis proteins displayed as 15-amino acid stretches. Inclusion/exclusion and significance analyses were performed according to published methods., Results: Each study group recognized 68-78% M. tuberculosis peptides at least once. M. tuberculosis epitope recognition by sarcoidosis patient sera was 42.7%, and by TB patient sera was 39.1%. Seven and 16 peptides were recognized in 9/12 (75%) and 8/12 (67%) sarcoidosis patient sera but not in TB patient sera, respectively. Nine (75%) and eight (67%) out of twelve TB patient sera, respectively recognized M. tuberculosis peptides that were not recognized in sarcoidosis patient sera., Conclusions: Specific IgG recognition patterns for M. tuberculosis antigens in sarcoidosis patients re-affirm mycobacterial involvement in sarcoidosis, providing biologically relevant targets for future studies pertaining to diagnostics and immunotherapy., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Bacille Calmette-Guérin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses., Methods: The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods., Results: IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor β (IL2Rβ), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFβR1) and, in agreement with the peptide microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFβR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rβ and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination., Conclusions: BCG vaccination modifies the host's immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Understanding of humoral immune responses in tuberculosis (TB) is gaining interest, since B-cells and antibodies may be important in diagnosis as well as protective immune responses. High-content peptide microarrays (HCPM) are highly precise and reliable for gauging specific antibody responses to pathogens, as well as autoantigens., Methods: An HCPM comprising epitopes spanning 154 proteins of Mycobacterium tuberculosis was used to gauge specific IgG antibody responses in sera of TB patients from Africa and South America. Open source software for general access to this method is provided., Results: The IgG response pattern of TB patients differs from that of healthy individuals, with the molecular complexity of the antigens influencing the strength of recognition. South American individuals with or without TB exhibited a generally stronger serum IgG response to the tested M. tuberculosis antigens compared to their African counterparts. Individual M. tuberculosis peptide targets were defined, segregating patients with TB from Africa versus those from South America., Conclusions: These data reveal the heterogeneity of epitope-dependent humoral immune responses in TB patients, partly due to geographical setting. These findings expose a new avenue for mining clinically meaningful vaccine targets, diagnostic tools, and the development of immunotherapeutics in TB disease management or prevention., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355)., Methods: Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes., Results: 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines., Conclusion: We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8(+) or CD4(+) T-cell response is to gauge the binding of candidate peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4(+) T-cell responses. We constructed a peptide microarray consisting of 7,466 unique peptides derived from 61 M. tuberculosis proteins. The peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual peptide species. Out of 7,466 peptides, 1,282, 674, and 1,854 peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained "hot spots," i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two peptides that formed MHC class II-peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these peptides serve as B-cell and CD4(+) T-cell epitopes. This work helps to identify not only M. tuberculosis peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.

Introduction: Peptide microarray slides usually contain positive control spots to gauge for antibody binding. Unlike the good response on earlier prototype microarrays, human immunoglobulin controls do not function consistently on newer generation slides. This may be due to technical problems in high-density printing or degradation. Our objective was to identify and print reliable control peptides that did not suffer from the same problems as proteins., Methods: Peptide microarray slides containing 10,000-23,000 synthetic peptides spanning proteins involved in M. tuberculosis or Bordatella pertussis were incubated with secondary antibody in sample dilution buffer. After removal of artefacts due to slide architecture, we identified peptides that gave a high mean response and low co-efficient of variation across all replicates. These control peptides were tested for their performance on newly manufactured slides., Results: We selected three peptides on the TB slides and three peptides on the B. pertussis slides that had a mean response index of at least 5 and a coefficient of variation less than 15%. When used as controls in newly-designed slides, these peptides gave consistently high responses: the median index ranged from 4.5 to 9.5 in the absence of patient serum and was of a somewhat lesser magnitude when incubated with patient serum. We illustrate the use of these control responses to normalize the peptide responses on a set of slides prepared with human serum., Conclusions: Our work shows that it is possible to identify control peptides that can be used when protein controls do not function consistently. This has important consequences for the storage of peptide-microarrays and their use in the field: protein chips need to be kept at +4 degrees C while peptide chips can be kept at room temperature. Although we focused on TB and B. pertussis, our methodology has relevance for any disease or disorder where peptide arrays are used to assess immune response.

Identification of major histocompatibility complex (MHC) class II binding peptides is a crucial step in rational vaccine design and immune monitoring. We designed a novel MHC class II molecule-peptide microarray binding assay and evaluated 346 peptides from already identified human immunodeficiency virus (HIV) epitopes and an additional set (n = 206) of 20-mer peptides, overlapping by 15 amino acid residues, from HIV type 1B (HIV-1B) gp160 and Nef as a paradigm. Peptides were attached via the N-terminal part to a linker that covalently binds to the epoxy glass slide. The 552 peptides were printed in triplicate on a single peptide microarray chip and tested for stable formation of MHC class II molecule-peptide complexes using recombinant soluble DRB1*0101(DR1), DRB1*1501(DR2), and DRB1*0401(DR4) molecules. Cluster analysis revealed unique patterns of peptide binding to all three, two, or a single MHC class II molecule. MHC class II binding peptides reside within previously described immunogenic regions of HIV gp160 and Nef, yet we could also identify new MHC class II binding peptides from gp160 and Nef. Peptide microarray chips allow the comprehensive and simultaneous screening of a high number of candidate peptide epitopes for MHC class II binding, guided by subsequent quality data extraction and binding pattern cluster analysis.

The data files produced by digitising peptide microarray images contain detailed information on the location, feature, response parameters and quality of each spot on each array. In this chapter, we will describe how such peptide microarray data can be read into the R statistical package and pre-processed in preparation for subsequent comparative or predictive analysis. We illustrate how the information in the data can be visualised using images and graphical displays that highlight the main features, enabling the quality of the data to be assessed and invalid data points to be identified and excluded. The log-ratio of the foreground to background signal is used as a response index. Negative control responses serve as a reference against which "detectable" responses can be defined, and slides incubated with only buffer and secondary antibody help identify false-positive responses from peptides. For peptides that have a detectable response on at least one subarray, and no false-positive response, we use linear mixed models to remove artefacts due to the arrays and their architecture. The resulting normalized responses provide the input data for further analysis.

Background: Serum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M. tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations., Method: We constructed a high-content peptide microarray with 61 M. tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/-., Findings: Three distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB-, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB- does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope 'hotspots' at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background., Conclusions: A uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize biologically relevant targets useful for development of novel TB diagnostics and vaccines.