Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Summary

Novel generations of functional assays such as gamma interferon (IFN-γ) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Abstract

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.  

Introduction

First performed in 1989, UCBT is increasingly used as part of the treatment of various neoplastic and nonneoplastic blood disorders in children¹. VZV is a cytopathic human alphaherpesvirus which causes two different diseases, varicella (after primary infection) and herpes zoster (after reactivation). Following primary infection, VZV persists throughout the life of the host sheltered within sensory nerves of dorsal root ganglia. One of the most threatening infectious complications following UCBT is associated with VZV^2-4. In our clinical center, in absence of VZV prophylaxis, the cumulative incidence of VZV disease VZV disease at 3 years postUCBT was 46%². In these patients, de novo infection with or reactivation of VZV is often associated with visceral dissemination to the central nervous system, lungs and liver^5-7. As a result, acyclovir, valacyclovir or famciclovir prophylaxis is commonly administrated to UBCT recipients^8,9. However, this treatment strategy does not take into account the protective potential of VZV specific T lymphocytes or the kinetics of reconstitution of VZV specific T cell responses. Potential problems associated with the expanding use of long term antiherpetic prophylaxis include a) patient overtreatment; b) the development of antiviral drug resistance^10,11; and c) impairment of VZV specific immune reconstitution^12,13. Because detection of functional VZV specific T lymphocytes correlates with the presence of long term protection from VZV infection and improved clinical outcome^4,14,15, monitoring cell mediated immune responses directed against VZV during the posttransplant period might result in a more rational use of antiviral treatment by enabling medical practitioners to distinguish patients who would benefit from VZV prophylaxis from those whose immune system is capable of controlling VZV replication^4,13.

The IFN-γ ELISpot assay is widely used for the monitoring cell mediated immune responses in a variety of experimental systems and clinical conditions. Spots are generated following the cleavage of a chromogenic substrate, generating a visible and stable precipitate at the site of the reaction. Each individual spot thereby represents the footprint of an individual cytokine-producing cell. IFN-γ ELISpot not only measures the ability of individual cells ex vivo to produce IFN-γ in response to in vitro stimulation with cognate antigen, but it also provides an estimate of the frequency of responding cells in a given cell population^16,17. In addition to its high sensitivity, IFN-γ ELISpot is straightforward to perform, making its use possible in the context of personalized clinical protocols aimed at guiding initiation or cessation of antiviral treatment. The procedure detailed below describes an ELISpot assay that is specifically designed to detect and measure the production of IFN-γ by peripheral blood mononuclear cells following in vitro stimulation with VZV derived antigens.

Protocol

This research protocol was approved by the Institutional Ethics Review Board of CHU Sainte-Justine, Montreal, Quebec, Canada, where the study was conducted. Informed consent was sought and obtained from all study participants, their parents or legal guardians. All procedures performed on days 1 and 2 must be carried out under sterile conditions (i.e. under a laminar flow hood). Standard safety procedures for handling human blood should be strictly observed.

1. Coating the Plates

1. Permeabilize the polyvinylidene difluoride (PVDF) membranes at the bottom of 96 well MultiScreen IP white ELISpot plates by adding to each well 20 μl of 35% ethanol for 1 min. Membranes should become slightly translucent.
2. Immediately wash the wells 3 times with 200 μl of 1x phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) using a multichannel pipette or similar device. This step is critical given that ethanol residues can affect cell viability and binding of capture antibody. ELISpot plates are fragile and should be handled carefully: the use of an automated plate washer is not recommended.
3. Coat the wells with 10 μl of purified mouse anti-human IFN-γ capture antibody diluted in 1x PBS to a final concentration of 10 μg/ml. Cover the plates with regular plastic wrap and incubate overnight at 4 °C.

2. Blocking Plates

1. Empty the wells, tapping them dry, and wash plates 5 times with 200 μl of 1x PBS.
2. Fill the wells with 200 μl of complete RPMI 1640 medium and incubate plates for 2 hr at 37 °C (blocking step).
3. Wash the plates 5x with 200 μl of 1x PBS and keep the wells full of 1x PBS until cells are ready to be plated.

3. Cell Plating and Stimulation

1. Prepare peripheral blood mononuclear cells (PBMC) from human venous blood samples by centrifugation on Ficoll-Paque gradients using standard procedures. Alternatively, using standard methods, thaw aliquots of PBMC frozen under liquid nitrogen. After the last centrifugation, resuspend cells at a final concentration of 2 x 10⁶ cells/ml and incubate overnight at 37 °C under a 5% CO₂ atmosphere in a water jacket incubator.
2. Remove PBMC from the incubator and resuspend them in a final volume of 5 ml of complete RPMI 1640 medium.
3. Incubate PBMC in the presence of 10 μl of genetically engineered endonuclease from Serratia marcescens for 5 min at room temperature.
4. Remove a 50 μl aliquot of PBMC and mix with 50 μl of trypan blue dye. Count cells and estimate % viability using a haemocytometer and microscopic examination (dye exclusion method). Various automated methods for cell counting and assessment of cell viability can also be used. PBMC should be >95% viable. Discard sample when viability is lower than 95%.
5. Centrifuge PBMC at 700 x g for 5 min, discard supernatant, and resuspend cells in AIM-V medium supplemented with 2% (v/v) inactivated human serum (HIS) at a final concentration of 2 x 10⁶ cells/ml. Keep at 37 °C until stimulation mixtures are added to the plate.
6. Add to the wells, one drop at a time, 100 μl of the appropriate stimulation mixture. Three stimulation mixtures should be used as described below:
   1. Use AIM-V medium supplemented with 2% (v/v) inactivated human serum (IHS) as negative control.
   2. Use anti-CD3 monoclonal antibody (clone OKT3) diluted in AIM-V medium supplemented with 2% (v/v) IHS to a final concentration of 0.5 μg/ml as positive control).
   3. Use VZV antigen, in the form of either γ-irradiated (10,000 Gy; 30 min exposure time) live attenuated VZV vaccine (1,350 plaque forming units [PFU]/ml) diluted 1:200 in AIM-V medium supplemented with 2% (v/v) IHS) or 1 μg of an equimolar mixture of 67 peptides (15 amino acid residues) synthesized based on the sequence of the VZV immediately early (IE63) phosphoprotein. Control irradiation efficacy by monitoring the absence of visible signs of cytopathic effects after 4 days in PBMC cultures.
   4. Prepare all wells in duplicate.
7. Add to the wells, one drop at a time, 100 μl of cells prepared in step 5.4 to the appropriate wells. Two wells should be left without cells (negative control). Incubate for 20 hr at 37 °C under a 5% CO₂ atmosphere in a water jacket incubator. Do not shake, move, or stack the plates on top of one another during incubation.

4. Spot Development and Detection

1. Discard the cells and wash the plates 10x with 1x PBS supplemented with 0.05% (v/v) Tween 20 (PBST). Tween 20 helps detach cells that have adhered to the PVDF membrane following overnight incubation.
2. Using a multichannel pipette, add 100 μl of 0.5 mg/ml biotin conjugated anti-IFN-γ monoclonal antibody (4S.B3 clone) diluted in 1x PBS containing 0.5% (w/v) bovine serum albumin (BSA). Incubate plates for 2 hr at room temperature.
3. Wash the wells 6x with PBST and add each well 100 μl of streptavidin conjugatd with alkaline phosphatase diluted 1:1,000 in 1x PBS containing 0.5% (w/v) BSA. Cover the plates and incubate for 1 hr at room temperature.
4. Wash the plates 3x with PBST and 3x with 1x PBS. Add 100 μl of a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium chloride (NBT) substrate solution and incubate 5 min at room temperature.
5. Wash the plates under running distilled water. Remove the bottom section of the ELISpot plate and wash both sides of the membrane with distilled water. Air dry the plates.
6. Examine the wells and enumerate the spots using a stereoscopic dissection microscope or scan the plates using an automated spot counter. Keep the plates away from light if they cannot be examined on the same day.
   1. Average the numbers of spots counted in corresponding duplicate wells and subtract the number of spots counted in the negative control wells (no cells) from the number of spots counted in the test wells.
   2. Express IFN-γ production as the number of spot-forming units (SFU) per 10⁶ PBMC.
   3. Consider that test samples are positive if the number of SFU is >50 per 10⁶ PBMC and 2 standard deviations (SD) above the negative control (cells + AIM-V medium supplemented with 2% (v/v) IHS).
   4. Use a value of 400 SFU per well when spots are too numerous to be counted.
7. Test whether ELISpot data are normally distributed or not using the Kolmogorov-Smirnov test. When data are normally distributed, perform statistical comparisons using Student’s t test (2 groups) or ANOVA and the Bonferroni post-test (multiple comparisons). If data are not normally distributed, use the Mann-Whitney U test (2 groups) or the Kruskal-Wallis test and Dunn’s post-test (multiple comparisons).

Results

The IFN-γ ELISpot protocol detailed above was developed and optimized in our laboratory to measure the magnitude and the quality of cell mediated immune responses directed against VZV⁴. Diverse sources of VZV antigen can be used for the stimulation step. These include: a) commercially available detergent inactivated extracts from VZV infected Vero cells¹⁸; b) pools of overlapping synthetic peptides from specific VZV encoded proteins, including IE63¹⁵ and ORF4¹⁹; c) live at...

Discussion

Modifications and troubleshooting: IFN-γ ELISpot assays have been used to examine cell-mediated immune responses directed against a variety of microbial pathogens, including human immunodeficiency virus type 1 (HIV-1)^24,25, hepatitis C virus (HCV)^26,27, and Mycobacterium tuberculosis^28,29, just to name a few. Here we described the development of an IFN-γ ELISpot assay to measure cellular immunity against, with the hope of defining correlates of VZV specific immune recon...

Materials

Name	Company	Catalog Number	Comments
Leucocep tube	VWR	89048-936/89048-932	12 ml or 50 ml tubes may be used depending on the volume of blood.
Ficoll-Paque	GE Healthcare	17-1440-02	Protect from light.
Benzonase nuclease	Novagen	70746-3	Keep at -20 C.
MultiScreenHTS-IP Filter Plate	Millipore	MSIPS4W10	Sterile with pore size of 0.45 µm.
Mouse anti-human IFN-γ capture antibody	BD Biosciences	551221	NIB42 clone.
Pepmix VZV IE63	JPT Peptide Technologies	PM-VZV-IE63	Dissolve contents of one vial in 40 μL of DMSO. Use within 6 months.
Biotin-conjugated anti-IFN-γ monoclonal antibody	BD Biosciences	554550	4SB3 clone.
Streptavidin conjugated with alkaline phosphatase	Bio-Rad Life Science	170-3554	Dilute for use on the same day.
BCIP/NBT	Bio-Rad Life Science	170-6432	Protect from light.