Detailed epitope mapping of humoral immune response towards persisting Epstein-Barr virus infections using peptide microarrays Paul von Hoegen, Nikolaus Pawlowski, Janina Seznec, Ulf Reimer JPT Peptide Technologies, Berlin, Germany. * Correspondence should be addressed to Ulf Reimer: [email protected] www.jpt.com Introduction All of us are infected by a number of viruses causing persistent infections which are staying in our bodies for a long period of time, sometimes the whole life. While, in most healthy individuals not recognized, persistent viral infections can cause a number of serious health problems. EBV, for instance, the causative agent of infectious mononucleosis infects over 90% of the human population. The EBV virus is thought to contribute to diseases such as different cancer types, multiple sclerosis and chronic fatigue syndrome. Additionally, persistent infections are a critical issue in organ and stem cell transplantation. The correlation of persistent infections with pathologies is an important issue; it is the gate to new diagnostics and therapies. However, passing the gate is still a demanding task. The detailed epitope mapping of humoral immune response in human serum samples allows a high resolution analysis of the antibody repertoire against EBV antigens. This information can then be correlated with clinical phenotypes. We developed a flexible peptide microarray platform which allows the presentation of up to 6900 peptides on a single microscope glass slide. Peptides are immobilized chemoselectively and directed onto the surface. A further highlight of the platform is the low volume of sample required; 1μl is enough. Serum incubations and data evaluation can be carried out using standard DNA- microarray equipment. We show examples where this approach allows to differentiate donors for anti EBV humoral immune responses. These results can be confirmed using a peptide ELISA approach. Library Design • Overlapping peptide scans through major EBV antigens (BLRF2, BZLF1, EBNA1, EBNA3, EBNA4, EBNA6, LMP1, VP26) (1465 peptides) • Follow-up library to include sequence variants and posttranslational modifications. Array Production Fig. 1. Schematic representation of the array production process. Fig. 2. Layout of peptide microarray (left) and images after printing (QC-scan, center) and after serum incubation (right). Three subarrays are used for improved data quality. Seroscreening • Screening of sera of healthy volunteers • Evaluation of images using GenePix • Processing of data and calculation for QC with R Data Quality Fig. 3. Typical intra array reproducibility between the three subarrays. Fig. 4. Patterns of antibody reactivities in human serum samples towards EBV antigens. Donor 1 shows antibody reactivity against EBNA1, donor 2 against EBNA1 and EBNA6 and donor 3 shows reactivity against almost all antigens except BLRF2 and EBNA6. Results • Individual signal patterns are observed for immunogenic proteins. • Signals span along the peptide scan Confirmation by Peptide ELISA Fig. 6. Serum reactivity towards individual peptides from EBNA1 measured by peptide microarray (left) and peptide ELISA (right). ELISA data are shown for two peptides (blue box in array data). Good correlation of signals is observed. Schematic layout QC-scan Image after incubation with serum SA1 SA2 SA3 Synthesis of peptide with N-terminal reactivity tag SA1 SA2 SA3 Fig. 5. Signal patterns for peptide scan through EBV capsid protein VP26 for three individuals. The shared residues in the overlapping peptide indicate the linear epitope recognized by antibodies of donor 3. • Development of a high content and high throughput screening platform for biomarker discovery • Screening of low volume serum samples results in specific signal patterns 1,2,3 • Signal patterns for persistent infection can be compared to clinical data • Library can be easily extended to sequence variants and posttranslational modifications. • Peptide microarray data can be efficiently confirmed and validated using peptide ELISA. VP26 SISSLRAATSGATAA VSSSISSLRAATSGA TPSVSSSISSLRAAT ALASSAPSTAVAQSA GTGALASSAPSTAVA ASAGTGALASSAPST QAAASAGTGALASSA References 1 Haynes et al.(2012) Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 366:1275-86. 2 Gaseitsiwe et al., (2010) Peptide microarray-based identification of Mycobacterium tuberculosis epitope binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401. Clin Vaccine Immunol. 17:168-75. 3 Maksimov et al., (2012) Analysis of clonal type-specific antibody reactions in Toxoplasma gondii seropositive humans from Germany by peptide-microarray. PLoS One. 7:e34212. Library generation Chemoselective immobilization on microarray slides Cleavage & reformatting SPOT- synthesis QC-Scan & post-printing procedures Printing process 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 NEG S01 S02 S03 POS AU PGRRPFFHPVGEADY FHPVGEADYFEYHQE S01 S02 S03 SSSSGSPPRRPPPGR SGSPPRRPPPGRRPF PPRRPPPGRRPFFHP RPPPGRRPFFHPVGE PGRRPFFHPVGEADY RPFFHPVGEADYFEY FHPVGEADYFEYHQE VGEADYFEYHQEGGP ADYFEYHQEGGPDGE FEYHQEGGPDGEPDV Peptide Array Data Peptide ELISA Data