Zika Virus Specific Diagnostic Epitope Discovery

Summary

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Abstract

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

Introduction

Zika virus (ZIKV) diagnosis based on clinical symptoms is challenging because it shares vectors, geographic distribution, and symptoms with Dengue and Chikungunya virus infection¹. Given the risk for adverse pregnancy outcomes in women infected with ZIKV during pregnancy, it is important to distinguish between the 3 viruses. Although the current molecular diagnostic tests are specific, they are only useful in blood or saliva during the relatively short period of acute infection^2,3. Serological assays are essential for diagnosis outside this initial period of infection⁴.

The development of a ZIKV specific serological assay is challenging for two reasons: first, the Zika antigens that the human immune system responds to are not currently known; and second, conserved flaviviruses amino acid sequences induce antibody cross-reactivity. Our objective was to discover unique ZIKV specific peptides to be used in diagnostics. Different approaches have been developed to screen peptide libraries covering entire proteins including phage, bacterial, and yeast surface display^5,6,7,8,9,10. Our strategy was to use a high-density peptide microarray that permits rapid and inexpensive high-throughput serological screenings^11,12 and subsequently the identified peptides can be used to improve current serological assays for detection of ZIKV infection.

This protocol enables the discovery of Zika virus specific diagnostic peptides using a high-density peptide microarray (Figure 1). The high-density peptide microarray was produced using the peptide laser printing technology. The entire ZIKV protein sequence consisting of 3,423 amino acid residues based on the French Polynesian strain (GenBank: KJ776791.2), was printed on a standard glass slide in blocks of 15 linear residues with an overlap of 14 amino acid residues in duplicate for a total of 6,846 peptide spots. In addition to the entire Zika protein sequence peptides, the microarray utilizes Influenza hemagglutinin (HA) peptides for internal controls.

A Zika validated positive serum sample obtained from Wadsworth Center (Albany, NY), was used to identify specific Immunoglobulin M (IgM) and Immunoglobulin G (IgG) reactive peptides. After incubating with the sample overnight, the microarray was stained with a secondary fluorochrome conjugated antibody (anti-human IgM or anti-human IgG), and analyzed on a microarray scanner. Quantification of spot intensities and peptide annotation was performed with a specific software provided by the same company that manufactured the microarray.

Protocol

These data are a part of an ongoing research study conducted at New York University College of Dentistry and were approved by the Institutional Review Board of the New York University School of Medicine, IRB # H10-01894. Clinical samples used in this study were de-identified samples used previously for diagnosis and with permission from the Wadsworth Center of New York State Department of Health, Albany, NY.

1. Installing the ZIKV High-density Peptide Microarray Slide in a specific Incubation Tray

1. Handle the glass slide by the edges using powder-free gloves. The glass slide dimensions are 75.4 mm by 25.0 mm and 1 mm of thick. Place the slide in the incubation tray with the microarray surface facing up.
2. Place the glossy side of the seal facing downwards onto the microarray printed surface. To ensure a good position, overlap the screw holes of the seal and the base plate.
3. Place the upper part of the tray onto the seal to create a microarray chamber.
4. Secure the slide in the tray by tightening equally the thumbscrews one after another by hand in an alternating pattern starting from the upper left followed by lower right. Place the lid of the incubation tray.

2. Background Interaction Detection: Staining the Microarray with Secondary Antibodies

NOTE: Use a pipette to deposit the solutions (standard and blocking buffers, diluted secondary antibodies) in the corner of the microarray chamber.

1. Incubate the microarray with a standard buffer (1x Phosphate buffered saline (PBS), 0.05% Tween 20, pH 7.4, filtered with a 0.45 µm filter) (total volume of 2,000 µL/microarray) for 15 min at room temperature (RT) on an orbital shaker at 140 rpm.
2. Remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber. Fill empty slide holders with blank slides to prevent breaking of the microarray slide.
3. Block the microarray with the blocking buffer (total volume of 2,000 µL/microarray) for 60 min at RT on an orbital shaker at 140 rpm.
4. Remove the blocking buffer by aspirating with a pipette from the corner of the microarray chamber.
5. Incubate the microarray with secondary antibody, anti-human IgM fluorochrome conjugated, diluted 1: 5,000 (total volume of 2,000 µL/microarray) in the staining buffer (10% blocking buffer in standard buffer) for 30 min at RT in the dark on an orbital shaker at 140 rpm.
6. Remove the secondary antibody by aspirating with a pipette from the corner of the microarray chamber.
7. Wash the microarray 3x, 1 min per wash with standard buffer (total volume of 2,000 µL/microarray at RT on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.
8. Immerse the slide 2x into a freshly prepared dipping buffer (1 mM Tris, pH 7.4), (total volume of 200 mL).
9. Dry the microarray carefully, for approximately 1 min, by aspirating excess fluid very carefully from the top to the bottom of the slide without touching the slide surface. Analyze the slide in a microarray scanner reader.

3. Exposure of the Microarray to Host Serum

CAUTION: Perform this step under laboratory safety conditions, Biosafety Level 2 (BSL-2) because of the potential infectious nature of the serum specimens. Work within a Class II biological safety cabinet (BSC).

1. Inactivate serum sample at 56 °C for 30 min and centrifuge at 16,000 rcf for 5 min at 4 °C¹³.
   NOTE: Use a pipette to deposit the solutions (staining, standard buffer and diluted serum) in the corner of the microarray chamber.
2. Dilute serum sample in staining buffer starting with a 1:1,000 (total volume of 2,000 μL/microarray) dilution. Keep it at 4 °C until use.
3. Incubate the microarray with the staining buffer (total volume of 2,000 μL/microarray) for 15 min at RT on an orbital shaker at 140 rpm.
4. Remove the staining buffer by aspirating with a pipette from the corner of the microarray chamber.
5. Incubate the microarray with the diluted serum sample overnight at 4 °C on an orbital shaker at 140 rpm.
6. Remove the diluted serum sample by aspirating with a pipette from the corner of the microarray chamber.
7. Wash the microarray 3x, 1 min per wash with standard buffer (total volume of 2,000 μL/microarray at RT on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.

4. Staining with Secondary Antibodies and Labeled Control Antibodies

NOTE: Use a pipette to deposit the solutions (standard buffers, diluted secondary and label antibodies) in the corner of the microarray chamber.

1. Dilute the secondary antibody in the staining buffer.
   NOTE: Use anti-human IgM fluorochrome conjugated antibody or anti-human IgG fluorochrome conjugated antibody at a dilution of 1:5,000 (total volume of 2,000 µL/microarray) in staining buffer.
2. Mix control label antibody (monoclonal anti-HA fluorochrome conjugated) at a dilution of 1:1,000 (total volume of 2 000 µL/microarray) in staining buffer with secondary antibody previously diluted in staining buffer.
3. Incubate with the microarray for 30 min at RT in the dark on an orbital shaker at 140 rpm.
4. Remove the mix of diluted secondary and label antibodies by aspirating with a pipette from the corner of the microarray chamber.
5. Wash 3x with standard buffer (total volume of 2,000 µL/microarray). Each wash is for 1 min on an orbital shaker at 140 rpm. After each wash, remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber.

5. Microarray Scanning

1. Immerse the slide 2x into freshly prepared dipping buffer (1 mM Tris, pH 7.4) (total volume of 200 mL).
2. Dry the microarray carefully, around 1 min, by aspirating very carefully from the top to the bottom of the slide without touching the slide surface.
3. Scan the microarray following the instructions of the scanner. Place the slide onto the scanner with the printed surface facing up.
4. Create a new project and select the scanning area. Set scanner parameters as: Resolution: 21 µm, Intensity for both 700 and 800 nm channels: 7.0, Scanning quality: medium and Offset: 0.8.
5. Acquire and save the scan raw image as a 16-bit grayscale TIFF file format.

6. Microarray Analysis Using a Specific Software

1. Open the raw image (TIFF image). Open the array grid file.
2. Align the array grid to the scan image with the computer's mouse or keyboard arrow keys.
3. Select "Quantify Selection" in the specific software, which creates a readout file that contains the signal intensity for each spot, the background value, and the corresponding peptide sequence.
   NOTE: This output file can also be imported into a spreadsheet program for additional analysis and graphing.

7. Microarray Storage

1. Store the microarray sealed in the dark at 4 °C under oxygen free nitrogen or argon gas.

Results

The results obtained using the protocol described are shown in Figure 2 and Figure 3. No background interactions were noted when pre-staining the microarray with secondary anti-human IgM (data not shown). Staining with an anti-human IgM conjugate resulted in several areas above background green fluorescence intensities, indicating the binding of these peptides with IgM in the host serum sample (

Discussion

We designed a protocol utilizing a high-density peptide microarray containing the entire Zika virus protein sequence (French Polynesian strain). The microarray was manufactured by printing 3,423 different overlapping linear peptides. Each peptide was 15 amino acids and varied by only one residue from its nearest neighbor in the sequence (i.e., 14 residue overlap). Although shorter overlaps can be printed, epitope mapping is more precise with longer overlaps. Each peptide was printed in duplicate to increase reli...

Materials

Name	Company	Catalog Number	Comments
PEPperCHIP Custom Peptide Microarray	PEPperPRINT	PPC.001.001	Custom peptide micarray: our microarray contains the entire Zika virus protein sequence
PEPperCHIP incubation tray 3/1	PEPperPRINT	PPC.004.001
PEPperCHIP Staining Kit (680 nm) (anti-HA, DyLight labeling)	PEPperPRINT	PPC.037.002
PepSLide Analyzer	PEPperPRINT	PSA.004.001	14-days free License for Windows
Rockland Blocking buffer	Rockland	MB-070
anti-human IgM (mu chain) DyLight 800	Rockland	609-145-007
anti-human IgG Fc DyLight 680	Thermo Scientific	SA5-10138
LI-COR Odyssey Imaging System	LI-COR
Orbital shaker device	IKA	MTS 2/4 digital microtiter shaker
Adobe Illustrator	Adobe
Deltagraph	Redrocks