Abstract Book 2010 - CIMT Annual Meeting

071 Kleemann | New targets & new leads
Identification of T-cell defined varicella-zoster virus proteins
Patrick Kleemann 1 , Stefan Tenzer 2 , Eva Distler 1 , Eva Wagner 1 Ralf G. Meyer 1 , Sebastian
Klobuch 1 , Simone Thomas 1 , Steffi Aue 3 , Bodo Plachter 3 , Wolfgang Herr 1
1 Dept. of Medicine III, Hematology & Oncology, University Medical Center, Mainz, Germany
2 Institute for Immunology, University Medical Center, Mainz, Germany
3 Institute for Virology, University Medical Center, Mainz, Germany
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Reactivated infection with varicella-zoster virus
(VZV) causes herpes zoster. In immunodeficient
cancer patients (e.g. after hematopoietic stem cell
transplantation) dissemination of the virus can lead
to life-threatening disease. Although an efficient
live attenuated VZV vaccine for zoster prophylaxis
exists, it is not approved in immunocompromised
patients due to safety reasons. Knowledge of immunogeneic
VZV proteins would allow designing a noninfectious
nonhazardous subunit vaccine suitable
for patients with immunodeficiencies. The objective
of this study is to identify T-cell defined virus
proteins of a VZV-infected Vero cell extract that we
have recently described as a reliable antigen format
for IFN-g ELISPOT assays (Distler et al. Biol. Blood
Marrow Transplant. 2008, 14:1417-24). We first
separated the VZV-infected/-uninfected Vero cell
extracts by ultracentrifugation and reverse-phase
HPLC. The collected fractions were screened for
reactivity with peripheral blood mononuclear cells
(PBMC) of VZV-seropositive healthy individuals
by IFN-g ELISPOT assay. Using this strategy, we
successfully identified bioactive HPLC fractions
that contained immunogeneic VZV material. The
immune reactivity was mediated by CD4+ memory
T cells of VZV-seropositive healthy individuals as
demonstrated by experiments with T-cell subpopulations.
We next analyzed the bioactive HPLC
fractions with MALDI and ESI mass spectrometry
techniques and identified three (glycoprotein
E; glycoprotein B and immediate early protein 62)
different VZV derived proteins. In subsequent experiments
we expressed these VZV-encoded proteins
in dendritic cells (DCs) by in vitro transcribed
RNA and screened transfected DCs with autologous
T cells. Based on these experiments we established
a protocol to perform these studies with PBMC of
healthy donors and patients after hematopoietic
stem cell transplantation. The work will hopefully
lead to protein candidates of VZV to be included
in a subunit vaccine, which can be safely used for
zoster prophylaxis in immunocompromised cancer
patients.