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: reimer@jpt.com

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 patterns1,2,3

• 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

EBNA1

S01

S02

S03

SSSSGSPPRRPPPGR

SGSPPRRPPPGRRPF

PPRRPPPGRRPFFHP

RPPPGRRPFFHPVGE

PGRRPFFHPVGEADY

RPFFHPVGEADYFEY

FHPVGEADYFEYHQE

VGEADYFEYHQEGGP

ADYFEYHQEGGPDGE

FEYHQEGGPDGEPDV

Peptide Array Data Peptide ELISA Data