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 ....

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....

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 attenua.......

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

List of materials used in this article

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.