Whole CMV proteome pattern recognition analysis after HSCT identifies unique epitope targets associated with the CMV status

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/- for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the 'exclusive recognition analysis (ERA)' to identify unique CMV epitope responses for each patient group. The 'exclusive recognition analysis' of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D-/R-). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution.The work has been funded by ALF (Arbetslivfonden) to M.M. and P.L. funds from Karolinska Institutet and Vinnova, Sweden to M.M

Pattern Recognition in Pulmonary Tuberculosis Defined by High Content Peptide Microarray Chip Analysis Representing 61 Proteins from M. tuberculosis

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+/2. 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 TB2, 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 TB2 does no

Validation of peptide epitope microarray experiments and extraction of quality data

Abstract. Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses

Major Histocompatibility Complex Class II Molecule-Human Immunodeficiency Virus Peptide Analysis Using a Microarray Chip▿ †

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

Intersection between PAM and the Exclusive Recognition analysis.

<p>The table lists peptides that were commonly defined by PAM (peptides with significant differences in the intensity of the response vs. the reference group D−R−) and as well in the ‘exclusive recognition analysis’ (never above a threshold for detection in the D−R− group). Peptides marked with a star (*) were above this threshold in all patients in the respective group but never in serum from D−R− patients. The average Q-value is the absolute difference between PAM Q-score for D−R− and the Q-score for each respective group. Higher average Q-value indicates the probability that the peptide is differently recognized between the respective group and the D−R− (reference) group.</p><p>At 6 months, possible cross-reactivity to serum IgG for the epitope QPGENEVRPHAGVID (HCMV UL102) and the aminoacid sequence from a DNA packaging tegument protein UL17 from <i>Herpes Simplex virus-1</i>, which shows a matching alignment of 5 amino acids without any mismatch. Note at 12 months, a possible cross-reactivity of serum IgG for the epitope AQLDLEADPTAREGE (HCMV UL35) and the aminoacid sequence from a protein from <i>Epstein-Barr virus</i>, RNGATFSKGDIEGNF (HCMV US30), the <i>Human herpesvirus-6A</i> protein, the epitope YPAVTTVYPPSSTAK and YDDESWRPLSTVDDH directed against proteins from <i>Human herpesvirus 8</i>, could be found. At 24 months, no matches (using the criteria outlined in materials and methods) between serum CMV epitope recognition and proteomes of other human herpesviruses were found.</p

Epitopes predicted by the Exclusive RECOGNITION analysis.

<p>The peptides presented are only detectable in serum from at least 4 out of 5 individuals in the respective group (D−R+, D+R− or D+R+, 6, 12 and 24 months post-HSCT) but never in the group D−R− at 6, 12, and 24 months post-HSCT.</p

Result of the microarray significant profiles analysis (MaSigPro).

<p><b>a</b>) Venn diagram with the number of significant peptides obtained in the three comparisons (each patient group vs. D−R−. The lists of peptides provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s009" target="_blank">Table S5</a> represents the entire set of peptides contained in the Venn diagram. These peptides were also grouped into 9 clusters (default value) according to their recognition profile (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s009" target="_blank">Table S5</a>). <b>b–c</b>) Cluster analyses using CMV peptides that were differentially recognized in serum from patients, based on the D−/R− status. Three representative peptide clusters are reported, one for each analysis: D−R+ vs. D−R− (top), D+R− vs. D−R− (middle), D+R+ vs. D−R− (bottom). b) The consistency of the CMV epitope response in the cluster is visualized using the continuous peptide recognition profile across all the samples. Each peptide in the cluster is represented with a different color. c) The group-averaged CMV epitope recognition profiles (for different time points after HSCT) are shown to visualize differences (between the different patient groups) for CMV peptides selected in each cluster. Each group is represented with a different color (red = D−R−, green = D−R+, blue = D+R−, cyan = D+R+). Below the figures, peptides in the three clusters are listed. All the identified clusters and peptides are reported in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s008" target="_blank">Table S4</a> in greater detail.</p

Intersection between PAM and the Exclusive Recognition analysis.

<p>The table lists peptides that were commonly defined by PAM (peptides with significant differences in the intensity of the response vs. the reference group D−R−) and as well in the ‘exclusive recognition analysis’ (never above a threshold for detection in the D−R− group). Peptides marked with a star (*) were above this threshold in all patients in the respective group but never in serum from D−R− patients. The average Q-value is the absolute difference between PAM Q-score for D−R− and the Q-score for each respective group. Higher average Q-value indicates the probability that the peptide is differently recognized between the respective group and the D−R− (reference) group.</p><p>At 6 months, possible cross-reactivity to serum IgG for the epitope QPGENEVRPHAGVID (HCMV UL102) and the aminoacid sequence from a DNA packaging tegument protein UL17 from <i>Herpes Simplex virus-1</i>, which shows a matching alignment of 5 amino acids without any mismatch. Note at 12 months, a possible cross-reactivity of serum IgG for the epitope AQLDLEADPTAREGE (HCMV UL35) and the aminoacid sequence from a protein from <i>Epstein-Barr virus</i>, RNGATFSKGDIEGNF (HCMV US30), the <i>Human herpesvirus-6A</i> protein, the epitope YPAVTTVYPPSSTAK and YDDESWRPLSTVDDH directed against proteins from <i>Human herpesvirus 8</i>, could be found. At 24 months, no matches (using the criteria outlined in materials and methods) between serum CMV epitope recognition and proteomes of other human herpesviruses were found.</p