The authors wish to thank study participants and their parents. We would also like to thank Dr. R&#233;jean Lapointe (CHUM Notre-Dame, Montreal, Canada) for access to his ELISpot reader, Dr. Lubo Alexandrov for statistical analysis, and Denis Blais, Sandra Caron, Silvie Valois and Martine Caty for expert technical assistance. Supported by grants from le Fonds d&#8217;op&#233;ration pour les projets de recherche clinique et d&#8217;&#233;valution des technologies (CHU Sainte-Justine) to H.S. and P.O., by la Fondation Centre de canc&#233;rologie Charles-Bruneau, and by the Leukemia &#38; Lymphoma Society of Canada. I.S.F. was supported by scholarships from la Fondation CHU Sainte-Justine and le Fonds de la recherche du Qu&#233;bec-sant&#233; (FRQS). A.J.G. was the recipient of scholarships from the Department of Microbiology, Infectiology &#38; Immunology, Universit&#233; de Montr&#233;al (Gabriel-Marquis Scholarship), FRQS, and the Canadian Institutes of Health Research (CIHR). N.M. was supported by la Fondation CHU Sainte-Justine, the Cole Foundation, and FRQS.

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
<ol...

Modifications and troubleshooting: IFN-&#947; 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 tuberculosis28,29, just to name a few. Here we described the development of an IFN-&#947; ELISpot assay to measure cellular immunity against, with the hope of defining correlates of VZV specific immune recon...

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 VZV2-4. 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 liver5-7. As a result, acyclovir, valacyclovir or famciclovir prophylaxis is commonly administrated to UBCT recipients8,9. 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 resistance10,11; and c) impairment of VZV specific immune reconstitution12,13. Because detection of functional VZV specific T lymphocytes correlates with the presence of long term protection from VZV infection and improved clinical outcome4,14,15, 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 replication4,13.

The IFN-&#947; 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-&#947; ELISpot not only measures the ability of individual cells ex vivo to produce IFN-&#947; 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 population16,17. In addition to its high sensitivity, IFN-&#947; 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-&#947; by peripheral blood mononuclear cells following in vitro stimulation with VZV derived antigens.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leucocep tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:143px;">89048-936/89048-932</td>
			<td style="width:397px;">12 ml or 50 ml tubes may be used depending on the volume of blood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ficoll-Paque</td>
			<td style="width:101px;">GE Healthcare</td>
			<td style="width:143px;">17-1440-02</td>
			<td>Protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzonase nuclease</td>
			<td style="width:101px;">Novagen</td>
			<td style="width:143px;">70746-3</td>
			<td>Keep at -20 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MultiScreenHTS-IP Filter Plate</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:143px;">MSIPS4W10</td>
			<td>Sterile with pore size of 0.45 &#181;m.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti-human IFN-&#947; capture antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">551221</td>
			<td>NIB42 clone.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pepmix VZV IE63&#160;</td>
			<td style="width:101px;">JPT Peptide Technologies</td>
			<td style="width:143px;">PM-VZV-IE63</td>
			<td>Dissolve contents of one vial in 40 &#956;L of DMSO. Use within 6 months.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-conjugated anti-IFN-&#947; monoclonal antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">554550</td>
			<td>4SB3 clone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated with alkaline phosphatase&#160;</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-3554</td>
			<td>Dilute for use on the same day.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BCIP/NBT</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-6432</td>
			<td>Protect from light.</td>
		</tr>
	</tbody>
</table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

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-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; 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. &#160;

The IFN-&#947; 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...

Watch this Scientific Journal Video about Development of an IFN-Î³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) 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).

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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Research

JoVE Journal

Immunology and Infection

15.5K visualizações.  Université de Montréal.  Novel gerações de ensaios funcionais, tais como o interferão gama (IFN-γ) ELISpot, que detectam a produção de citoquinas ao nível de células individuais e fornecer tanto a caracterização quantitativa e qualitativa das respostas das células T pode ser utilizado para avaliar as respostas imunes mediadas por células contra a varicela zoster vírus (VZV). 

Vídeo: Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation