AbstractBackground After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation.Design and Methods Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4+ and CD8+ memory T cells.Results Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4+ T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4+ and CD8+ T cells.Conclusions Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4+ and CD8+ T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.
Herpes zoster represents the clinical manifestation of reactivated varicella-zoster virus (VZV) infection and occurs with an incidence of 25–40% after allogeneic hematopoietic stem cell transplantation (HSCT).1–3 Although long-term antiviral drug prophylaxis with acyclovir and its derivatives is very effective in preventing zoster following transplantation, it does not provide complete protection and can postpone the disease to the late post-HSCT period.4–8 In addition, the success of antiviral prophylaxis can be compromised by lack of patient compliance or by renal dysfunction. In fact, renal dysfunction is quite common after transplantation and may require the discontinuation of prophylactic medication due to nephro-toxic side effects. Long-term application of antiviral agents may also favor the selection of drug-resistant virus mutants.9,10 A more causal approach for zoster prevention is to boost VZV-specific cellular immunity by vaccination to levels that are sufficient to protect transplant recipients from the disease. However, live attenuated vaccines that are successfully used for varicella and zoster prophylaxis in immunocompetent individuals are not approved in immunocompromised HSCT patients due to safety concerns.11–15
VZV vaccines suitable for HSCT recipients16 should be completely devoid of infectious virus. A candidate vaccine based on a heat-inactivated whole virus preparation of VZV has already demonstrated safety along with clinical and immunological activity in 2 pilot trials in the setting of HSCT.17,18 Since effective antiviral control upon transplantation mainly depends on the successful reconstitution of virus-specific T cells,1,19,20–22 VZV vaccines should contain immunodominant T-cell targets of VZV. However, there is a paucity of data on the nature of VZV antigens that drive posttransplant T-cell immunity and that are of sufficient immunostimulatory activity to protect HSCT recipients from the disease. A considerable number of glycoproteins and immediate early (IE) proteins of VZV have been identified as a source of CD4 and CD8 T-cell antigens.23 Amongst those, glycoprotein E (gE), IE62, and IE63 were defined as being immunodominant.24–28 The screening approaches that led to these findings mainly used memory T-cell populations which were expanded from PBMC of latently infected healthy individuals by antigen-specific stimulation over a culture period of several days to weeks. However, prolonged culturing may favor the proliferation of certain T-cell specificities over others and, consequently, the observed profile may not necessarily reflect the hierarchy of immunogenicity of different VZV antigens in vivo.
In order to match the in vivo situation as closely as possible and avoid in vitro bias, we established a novel screening approach. For this, VZV proteins derived from virus-infected cells were fractionated by reverse-phase high performance liquid chromatography (RP-HPLC). Individual fractions containing VZV proteins were subsequently incubated with PBMC from latently infected healthy donors in sensitive interferon (IFN)-γ ELISpot assays to stimulate antiviral memory CD4 and CD8 T lymphocytes directly ex vivo. Reactive fractions were further analyzed to identify individual VZV proteins by using concerted chromatography and mass spectrometry procedures. For verification, identified candidate antigens were tested as full-length recombinant proteins for recognition by T cells. T-cell stimulation assays were performed both with cells from latently infected healthy individuals and from patients with zoster after allogeneic HSCT, again restricting responder cell populations to ex vivo PBMC. Together these analyses confirmed VZV gE and IE62 were immunodominant T-cell antigens. Interestingly, the VZV glycoprotein B (gB) was identified as an additional major T-cell target. We further demonstrated that allogeneic HSCT patients develop strong in vivo expansion of CD4 and CD8 T cells targeting glycoproteins B and E during the onset of herpes zoster. Therefore, our data suggest that both glycoproteins are top candidates for the design of subunit VZV vaccines in the setting of HSCT.
Design and Methods
Donors and patients
The study was approved by the local ethics committee and was performed according to the Declaration of Helsinki. Informed consent was obtained from all participants. Healthy donors (HD) were VZV-immune volunteers (n=11). They provided whole blood donations used to isolate PBMC by buffy coat separation and subsequent Ficoll centrifugation. PBMC were stored frozen in liquid nitrogen until use. VZV-seropositive study patients (n=7) were treated with reduced-intensity allogeneic HSCT for acute or chronic leukemia. During conditioning therapy, 4 of them had received the lymphocyte-depleting antibody alemtuzumab, while T/B cell depletion agents had not been given to the other 3 patients. Anti-zoster drug prophylaxis with famciclovir was performed until day +365 after HSCT, unless toxic renal dysfunction prevented treatment. Patients developed localized herpes zoster at a median of +337 (range 90–935) days after HSCT. The disease was diagnosed clinically and was confirmed by VZV-specific PCR. Patients with acute zoster were treated with intravenous acyclovir over 10–14 days, followed by secondary prophylaxis with famciclovir. Patient PBMC were collected and cryopreserved before zoster, at zoster onset and at indicated time points thereafter. Serum samples of patients were analyzed for VZV IgG/IgM by ELISA (Virion-Serion, Würzburg, Germany). Stem cell donors were HLA-matched sibling (n=1), HLA-matched unrelated (n=4), or HLA-mismatched unrelated individuals (n=2). PBMC of stem cell donors were isolated from excess donor lymphocyte infusion material, if available. Mature dendritic cells (DC) were generated in vitro from peripheral blood monocytes as described.29
Biochemical purification of VZV-infected Vero cell lysate by reverse-phase HPLC
A lysate (0.4–0.7 mg/mL) prepared from VZV-infected Vero cells (Advanced Biotechnologies, Columbia, MD, USA) was used as antigen source. According to information provided by the manufacturer, cells had been infected with the VZV ROD strain for 7–9 days. A lysate prepared from uninfected Vero cells (Advanced Biotechnologies) by the same procedure was included as negative control. After 0.2 μm filtration, lysate filtrate was separated by RP-HPLC. RP-HPLC was performed with an Ettan LC system (GE Healthcare, Waukesha, WI, USA) equipped with a Jupiter 4 μm, C12, Proteo 90 Ǻ, (250 x 2.0 mm) column (Phemomenex Inc, Aschaffenburg, Germany). Then 50 μL of 0.2 μm filtrate were injected and separated with an acetonitrile gradient (5–25% over 10 min, 25–65% over 40 min, 65–90% over 10 min). The aqueous and organic mobile phases contained 0.1% trifluoroacetic acid in H2O and in acetonitrile, respectively. Fractions (each 150 μL) were collected at a flow rate of 150 μL/min and were split for subsequent use in bioassays and mass spectrometry (MS) analyses.
IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay
ELISpot assays were performed as previously described29 with minor modifications. PBMC, either loaded with VZV-infected cell lysate or HPLC fractions, or electroporated with RNA coding for VZV proteins, were incubated in antibody-coated ELISpot plates at 0.5–2×10 cells/well over 40 h to allow for processing, presentation, and immune recognition of VZV proteins. In experiments analyzing HLA restriction of T-cell reactivity, the following murine mAb were added to ELISpot wells at saturating concentrations: W6/32, an anti-HLA class I IgG2a;30 L243, an anti-HLA-DR IgG2a;31 SPV-L3, an anti-HLA-DQ IgG2a;32 and B7.21, an anti-HLA-DP IgG3.33
Protein identification by electrospray ionization mass spectrometry (ESI-MS)
RP-HPLC fractions (each 100 μL) were dried and re-solubilized in 25 mM ammonium bicarbonate containing 0.1% Rapigest (Waters, Eschborn, Germany). Solubilized proteins were subjected to reduction, alkylation and tryptic digestion as previously described.34 After removal of detergent by acid hydrolysis and centrifugation,34 the supernatant was transferred into an autosampler vial. Capillary liquid chromatography of tryptic peptides (2.6 μL injection) was performed with a Waters NanoAcquity UPLC system online coupled to a Waters Q-TOF Premier system as described.34 Raw data processing and database searching were performed as detailed35 with the IDENTITY Algorithm of ProteinLynx Global Server (version 2.3), using an in-house compiled database containing Uni-ProtKB/Swiss-Prot Protein Knowledgebase (http://expasy.org/sprot/) entries for macaca mulatta (349 entries), cercopithecus aethiops (181 entries), pongo pygmaeus (239 entries), homo sapiens (20,405 entries), pan troglodytes (688 entries), pan paniscus (122 entries), gorilla gorilla (279 entries), papio papio (190 entries), varicella-zoster virus (Dumas strain, 69 entries), supplemented with known possible contaminants (porcine trypsin). Maximum mass deviation was set to 15 ppm for precursor ions and 30 ppm for fragment ions. For valid protein identification at least two peptides had to be detected with altogether at least seven fragments. The false-positive rate for protein identification was set to 1% based on a search of a 5x-randomized database.
Production and electroporation of IVT-RNA
For in vitro transcription (IVT) of RNA coding for single VZV proteins, pcDNA™3.1-gE, pcDNA™3.1-gB, and pcDNA™3.1-IE62 vectors were used as DNA templates. The pcDNA™3.1 vectors encoding VZV proteins were kindly provided by Dr AM Arvin and Dr M Sommer (Stanford University, Stanford, CA, USA). IVT was performed with T7 RNA polymerase using the mMESSAGE mMACHINE T7 Ultra kit (Ambion/Applied Biosystems, Darmstadt, Germany). After enzymatic DNaseI digestion to remove the template DNA and subsequent enzymatic polyadenylation, the IVT-RNA was purified by the RNeasy Mini Kit (Qiagen, Hilden, Germany). PBMC or mature DC were then adjusted in OptiMEM medium (Gibco/Invitrogen, Darmstadt, Germany) to 2.5–10×10 per 200 μL and were transferred with 20 μg IVT-RNA in 4 mm electroporation cuvettes (Peqlab, Erlangen, Germany). Electroporation was performed with the GenePulser Xcell system (Bio-Rad, Munich, Germany) applying a square wave pulse of 350V/12 ms. Electroporated cells were cultured for 4 h at 37°C and were thereafter used in ELISpot assays or cLSM analysis.
Confocal laser scanning microscopy (cLSM)
Intracellular expression of VZV proteins was determined with a confocal Zeiss LSM 510-UV device equipped with LSM Image Examiner software (Zeiss, Jena, Germany). Cells were intracellularly stained (Cytofix/Cytoperm, BD Biosciences, Heidelberg Germany) for 30 min at 4°C with VZV protein-specific murine IgG1 mAb (Novus Biologicals, Littleton, USA) and FITC-conjugated goat-anti-mouse IgG (Immunotech, Marseille, France). Nuclear co-staining was performed by 200 nM Hoechst 33342 (Invitrogen). Subsequently, 10 cells were transferred into Lab-Tek 8-well chamber slides at 300 μL medium/well for imaging.
VZV-infected cell lysate contains T-cell antigens of VZV
In a first set of experiments, a VZV-infected Vero cell lysate was used as source of VZV antigens. After 0.2 μm filtration of the lysate, filtrate and retentate were analyzed for immune reactivity in PBMC from healthy individuals with latent VZV infection in IFN-γ ELISpot assay. Cytokine secretion was significantly increased when the filtrate compared to the retentate was incubated with PBMC (Online Supplementary Figure S1). Recognition was specific, as IFN-γ secretion was not detected using the uninfected Vero cell lysate preparation as control. The immunogenic filtrate material was subsequently separated on a C12 column by RP-HPLC. Aliquots of all fractions were analyzed for immune recognition, again by using PBMC of VZV-immune healthy donors as screening populations in IFN-γ ELISpot assay. Distinct reactivity to several RP-HPLC fractions was found, indicating that they contained immunore-active VZV antigens (Figure 1). The pattern of reactive and non-reactive fractions was consistent in 3 of 3 analyzed individuals. Subsequent ELISpot assays with purified T-cell subsets (i.e. CD4, CD8) and autologous dendritic cells showed that reactivity to fractions was predominantly mediated by CD4 T cells (data not shown).
Mass spectrometry identification of VZV-encoded proteins
To identify immunoreactive VZV antigens, we performed quantitative mass spectrometric analysis (qMS) of RP-HPLC fractions. Proteins contained in those fractions that stimulated T-cell activation were first subjected to tryptic digestion. Resulting peptides were then analyzed by nanoUPLC-ESI-Q-TOF mass spectrometry. Acquired spectra were processed with ProteinLynx Global Server (PLGS 2.3), followed by a search in a composite database containing VZV and primate protein sequences. With the use of this approach, the VZV-encoded glycoproteins gB and gE as well as the immediate early protein 62 (IE62) were detected in immunoreactive HPLC fractions (Figures 2A-C). These viral proteins were not found in non-reactive fractions. The latter contained several other proteins, including VZV glycoprotein I (data not shown). Additionally, the relative abundance of gB, gE, and IE62 protein fragments in individual HPLC fractions as determined by qMS correlated well with the magnitude of reactivity of virus-immune donors in IFN-γ ELISpot assay (Figure 2D). Taken together, these data suggested viral gB, gE, and IE62 as major target proteins of the natural human T-cell response during the latency stage of VZV infection.
VZV gB, gE and IE62 are major targets of CD4+ T cells in latently infected individuals
To confirm our initial findings and to define T-cell populations directed against individual VZV proteins, cDNA clones encoding full-length viral gB, gE, and IE62 were transcribed in vitro (IVT) into RNAs. The latter were subsequently transfected into PBMC by electroporation. Confocal LSM on electroporated DC (Figure 3) and PBMC verified expression of the viral proteins, with gB and gE being located in the cytoplasm and on the cell membrane and IE62 being mainly detectable in the nucleus of transfected cells (data not shown). Transfected PBMC were then analyzed for IFN-γ secretion by T cells in 10 healthy virus carriers. Clear recognition of 2 to 3 virus proteins was observed in every single donor tested (Figure 4A and B). The reactivity was detectable over a broad RNA dose range (1–30 μg) and reached peak intensities at 20 μg (data not shown). Median spot numbers per 10 PBMC, induced by glycoproteins B (87, 21-495) and E (83, 17-511) exceeded that induced by IE62 (49, 1-237), respectively (Figure 4C). By using mAb that block interaction of the T-cell receptor with either HLA class I or II molecules, VZV protein reactivity of healthy individuals was found to be mainly mediated by HLA class II restricted T cells. In contrast, T cells recognizing these three VZV proteins in association with HLA class I were detected at much lower numbers (Figure 4D). These data suggested that immune reactivity to VZV proteins in healthy virus carriers is predominantly mediated by virus-specific CD4 T cells, which was confirmed in additional assays using purified T-cell subsets and DC (Online Supplementary Figure S2).
Posttransplant zoster triggers strong CD4+ and CD8+ T-cell responses to VZV gB and gE
The specificity of VZV-specific T cells was subsequently analyzed during the course of localized herpes zoster after allogeneic HSCT. Serial PBMC samples from zoster patients (n=5) were transfected with individual IVT-RNAs and tested in IFN-γ ELISpot assays. Vigorous expansions of T cells recognizing VZV gB and gE were observed after zoster onset, while IE62 reactivity was negative or at a very low level (Figure 5). In contrast, T-cell responses to any of these 3 VZV proteins were not detected in PBMC obtained from these patients before zoster occurred. However, T cells with reactivity to VZV proteins were clearly found in 3 of 4 stem cell donors prior to transplantation (Figure 5). In patients (n=7) analyzed during the first four weeks after zoster onset, median IFN-γ spot numbers per 10 PBMC to glycoproteins B (36, range 0–177) and E (47, range 8–323) exceeded that to IE62 (0, range 0–5), respectively (Figure 6A). Anti-HLA blocking assays suggested that this early glycoprotein reactivity after zoster was mediated by both CD4 and CD8 T cells (Figure 6C). Additionally, we observed that the frequency of circulating gE-specific T cells exceeded that of gB during the second month after zoster (Figure 6B) and that the level of VZV cell lysate reactivity was either below, similar or above that of total reactivity to all 3 VZV proteins (Figures 5 and 6). In contrast to mixed CD4 and CD8 T-cell responses to electroporated viral glycoproteins, reactivity to the VZV cell lysate was mediated only by CD4 T cells (Figures 6D). Clinical follow up of the 7 analyzed patients showed that none of them developed a second episode of zoster during a median period of 25 (range 9–36) months after first occurrence. Together these experiments indicated that herpes zoster occurring after allogeneic HSCT triggers marked CD4 and CD8 T-cell responses to VZV gE and gB.
Here we combined RP-HPLC fractionation of a virus-infected cell lysate with sensitive IFN-γ ELISpot assay and quantitative mass spectrometry to define T-cell antigens of VZV for subsequent immunogenicity analyses in latently infected donors and in allogeneic HSCT patients with zoster. Besides viral glycoprotein E and immediate early protein IE62, which had been reported before as major T-cell targets,23,26,27 glycoprotein B was identified as an additional immunodominant VZV protein inducing both CD4 and CD8 T-cell reactivity. Our approach has considerable advantages over many other methods in identifying viral T-cell antigens. It allows for the rapid detection of naturally expressed candidate antigens from a relatively low (<1 mg) initial amount of virus-infected cell lysate material. Furthermore, the use of PBMC from VZV-seropositive donors as the preferred screening population in a sensitive IFN-γ ELISpot assay avoids in vitro culturing of T cells. The latter is both time-consuming and prone to bias due to possible selective expansion of distinct T-cell specificities under the chosen culture conditions. In addition, our approach is extremely flexible. First, screening with PBMC from patients with active herpes zoster may be suitable to help us unravel additional T-cell antigens of VZV that could play an important role during the acute phase of the disease. Moreover, the virus antigen source used in this study was prepared from Vero cells after one week of VZV infection. Modification of the infection period with regard to time and addition of drugs interfering with virus metabolism could most likely change the composition and concentration of expressed virus proteins and might lead to the identification of additional known (e.g. glycoprotein I, IE63)23 or even novel T-cell antigens of VZV. It is well conceivable that the system can be extended towards other viruses in which T-cell antigens are completely or partly undefined.
For verification, VZV candidate proteins identified by qMS were expressed in PBMC by electroporation of full-length IVT-RNAs. This procedure enabled the in vitro stimulation and detection of a broad repertoire of VZV-specific T cells, bypassing the requirement for prior analysis of HLA type and HLA allele-specific peptide epitopes of the test person. In contrast to loading target cells with purified proteins or cell lysates, protein expression after RNA transfection not only allows peptide presentation by MHC class II, but also efficiently generates epitopes presented by the MHC class I pathway.36 Using this approach in combination with HLA-A/B/C- and HLA-DR/DQ/DP-blocking antibodies we demonstrated that virtually every latently infected healthy donor carried significant numbers of circulating CD4 T cells directed against at least 2 of the 3 VZV proteins. In contrast, CD8 T cells recognizing the same target proteins were detected at lower numbers in virus-immune healthy individuals. Considering the fact that in every assay the entire VZV lysate or protein reactivity could be completely blocked by HLA antibodies (of either class I or II specificity) indicated recognition by MHC restricted T cells and not by non-MHC restricted effector cells. Our observation is in accordance with previous reports which had shown a predominance of VZV-specific CD4 memory T cells during latent infection.23,26 We further found that the precursor frequencies of circulating VZV IE62-reactive T cells were much higher than previously reported.27,37 In contrast, frequencies of gE-specific T cells were consistent with previous studies, using overlapping peptides and the IFN-γ ELISpot assay for monitoring.38 Surprisingly, however, the highest median numbers of peripheral blood T cells were found to be targeting gB, a protein that was previously much less appreciated as a source of antigenic peptides. This clearly demonstrates that gB is an important target of the T-cell response during VZV latency.
We monitored the frequencies of VZV protein-reactive T cells in seropositive patients who developed localized herpes zoster after reduced-intensity allogeneic HSCT. Before onset of zoster symptoms, the numbers of circulating protein-reactive T cells were below the detection limit of the assay (<5 per 10 PBMC), even in those patients who did not receive T-cell depleting agents during conditioning therapy. These findings matched the lack of IFN-γ ELISpot responses to VZV-infected cell lysate which we had reported for pre-zoster PBMC samples of 12 VZV-seropositive patients undergoing T-cell depleted allogeneic HSCT.21 The frequencies of T cells specifically recognizing VZV glyco-proteins B and E, however, increased considerably during the first four weeks after zoster onset, whereas that to IE62 remained negative or reached only a very low level. The reasons for the lack of detectable IE62-specific T cells in our zoster patients is still unclear. Interestingly, we could show that substantial numbers of HLA class I restricted gB and gE reactive CD8 T cells are present during the first weeks after zoster onset, which most likely assisted antiviral CD4 T cells23,26,27 in the total cytolytic T-cell response. Supporting evidence for this hypothesis would require the detection of cytolysis-associated molecules (e.g. perforin, granzyme B) in PBMC, which was not possible in the current study due to limited sample material. The levels of IFN-γ ELISpot reactivity to gB and gE were followed for several months and reached almost those found in healthy virus-immune donors.
The described assay system can be implemented in similar monitoring studies39 in patients in other situations of immunodeficiency (e.g. autologous HSCT, cancer, HIV, the elderly), where the incidence of herpes zoster is also significantly increased. These analyses might confirm the observed predominant role of gB and gE for T-cell mediated immunity to VZV and/or define other important T-cell antigens during zoster. Interestingly, we found in several PBMC samples that the sum of reactivity to single VZV proteins was higher compared to that to the entire VZV cell lysate (e.g. patients 2 and 5). Thus, the use of viral cell lysates as the antigen source in monitoring assays may underestimate total anti-VZV reactivity, because lysates usually contain relatively low concentrations of VZV proteins and favor the stimulation of antiviral CD4 (and not CD8) responses. In other PBMC samples, however, the level of VZV cell lysate reactivity was clearly above that of total reactivity to all 3 VZV proteins (e.g. patients 3 and 4). This observation indicated that alternate targets beyond those studied here can dominate the T-cell response to VZV.
Live attenuated VZV vaccines are suitable to effectively enhance antiviral T-cell immunity in immunocompetent individuals.40,41 It has also been shown in 2 randomized studies that a heat-inactivated formulation of a live attenuated VZV vaccine can boost VZV-reactive CD4 T cells in patients undergoing autologous and allogeneic HSCT, and that this specific reconstitution correlates with protection from zoster.17,18 We show here that VZV gB and gE are major targets of virus-specific CD4 and CD8 T-cell reconstitution occurring during zoster after allogeneic HSCT. None of the patients analyzed developed a second episode of zoster during a median period of 25 months after first occurrence. Although this observation does not prove a direct causative role of anti-glycoprotein reactivity for zoster prevention, VZV gB and gE may form the basis for a safe zoster subunit vaccine, suitable for vaccinating both donors and recipients of HSCT. Studies with gE subunit vaccines already demonstrated the induction of specific B and T-cell responses in animals.42,43 Interestingly, preliminary results from an open randomized study in adults exploring a recombinant gE vaccine with adjuvant for zoster prevention demonstrated more robust cellular (i.e. CD4) and humoral immune responses in older adults compared to the application of a live varicella vaccine.44 Clinical activity and adverse effect profile in ongoing trials as well as those to be performed in HSCT patients will show if VZV glycoprotein subunit vaccines are an attractive alternative compared to whole virus vaccines. Considering that B-cell immunity is not suitable for preventing zoster after allogeneic HSCT,1 stimulation of VZV-specific T cells will be a major goal in developing an efficient vaccine for HSCT patients. Monitoring such T-cell responses on the basis of individual VZV proteins in vaccinated subjects could be an important application for the described assay system. In prospective trials, it may help to optimize vaccine strategies and to define a threshold frequency of circulating VZV antigen-specific T cells that correlates with protection from zoster and may allow for safe discontinuation of antiviral drug prophylaxis.
The authors would like to thank Dr Ann M Arvin and Dr Marvin Sommer, Departments of Pediatrics, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, CA, USA, for providing pcDNA™3.1 vectors that encode VZV proteins.
- Funding: this work was supported by a grant from the Deutsche José Carreras Leukämie Stiftung to W.H. B.P. was supported by the KFO183 program grant of the DFG. St.T. and H.S. were supported by the Immunology Research Center of the University Medical Center, Mainz and the DFG (SFB490, Z3).
- The online version of this article has a Supplementary Appendix.
- Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
- Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.
- Received August 2, 2011.
- Revision received November 17, 2011.
- Accepted December 19, 2011.
- Arvin AM. Varicella-Zoster virus: pathogenesis, immunity, and clinical management in hematopoietic cell transplant recipients. Biol Blood Marrow Transplant. 2000; 6(3):219-30. PubMedhttps://doi.org/10.1016/S1083-8791(00)70004-8Google Scholar
- Han CS, Miller W, Haake R, Weisdorf D. Varicella zoster infection after bone marrow transplantation: incidence, risk factors and complications. Bone Marrow Transplant. 1994; 13(3):277-83. PubMedGoogle Scholar
- Koc Y, Miller KB, Schenkein DP, Griffith J, Akhtar M, DesJardin J, Snydmann DR. Varicella zoster virus infections following allogeneic bone marrow transplantation: frequency, risk factors, and clinical outcome. Biol Blood Marrow Transplant. 2000; 6(1):44-9. PubMedhttps://doi.org/10.1016/S1083-8791(00)70051-6Google Scholar
- Ljungman P, Wilczek H, Gahrton G, Gustavsson A, Lundgren G, Lonnqvist B. Long-term acyclovir prophylaxis in bone marrow transplant recipients and lymphocyte proliferation responses to herpes virus antigens in vitro. Bone Marrow Transplant. 1986; 1(2):185-92. PubMedGoogle Scholar
- Ljungman P. Prophylaxis against her-pesvirus infections in transplant recipients. Drugs. 2001; 61(2):187-96. PubMedhttps://doi.org/10.2165/00003495-200161020-00004Google Scholar
- Thomson KJ, Hart DP, Banerjee L, Ward KN, Peggs KS, Mackinnon S. The effect of low-dose aciclovir on reactivation of varicella zoster virus after allogeneic haemopoietic stem cell transplantation. Bone Marrow Transplant. 2005; 35(11):1065-9. PubMedhttps://doi.org/10.1038/sj.bmt.1704959Google Scholar
- Boeckh M, Kim HW, Flowers ME, Meyers JD, Bowden RA. Long-term acyclovir for prevention of varicella zoster virus disease after allogeneic hematopoietic cell transplantation--a randomized double-blind placebo-controlled study. Blood. 2006; 107(5):1800-5. PubMedhttps://doi.org/10.1182/blood-2005-09-3624Google Scholar
- Erard V, Guthrie KA, Varley C, Heugel J, Wald A, Flowers ME. One-year acyclovir prophylaxis for preventing varicella-zoster virus disease after hematopoietic cell transplantation: no evidence of rebound varicella-zoster virus disease after drug discontinuation. Blood. 2007; 110(8):3071-7. PubMedhttps://doi.org/10.1182/blood-2007-03-077644Google Scholar
- Reusser P, Cordonnier C, Einsele H, Engelhard D, Link D, Locasciulli A, Ljungman P. European survey of herpesvirus resistance to antiviral drugs in bone marrow transplant recipients. Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant. 1996; 17(5):813-7. PubMedGoogle Scholar
- Villarreal EC. Current and potential therapies for the treatment of herpes-virus infections. Prog Drug Res. 2003; 60:263-307. PubMedGoogle Scholar
- Hardy I, Gershon AA, Steinberg SP, LaRussa P. The incidence of zoster after immunization with live attenuated varicella vaccine. A study in children with leukemia. Varicella Vaccine Collaborative Study Group. N Engl J Med. 1991; 325(22):1545-50. PubMedhttps://doi.org/10.1056/NEJM199111283252204Google Scholar
- Vazquez M, LaRussa PS, Gershon AA, Steinberg SP, Freudigman K, Shapiro ED. The effectiveness of the varicella vaccine in clinical practice. N Engl J Med. 2001; 344(13):955-60. PubMedhttps://doi.org/10.1056/NEJM200103293441302Google Scholar
- Oxman MN, Levin MJ, Johnson GR, Schmader KE, Straus SE, Gelb LD. A vaccine to prevent herpes zoster and pos-therpetic neuralgia in older adults. N Engl J Med. 2005; 352(22):2271-84. PubMedhttps://doi.org/10.1056/NEJMoa051016Google Scholar
- LaRussa P, Steinberg S, Meurice F, Gershon A. Transmission of vaccine strain varicella-zoster virus from a healthy adult with vaccine-associated rash to susceptible household contacts. J Infect Dis. 1997; 176(4):1072-5. PubMedhttps://doi.org/10.1086/516514Google Scholar
- Patel SR, Ortin M. Varicella-zoster reactivation in a patient receiving routine revaccinations after an allogeneic hemopoietic progenitors transplant. J Pediatr Hematol Oncol. 2005; 27(2):106-8. PubMedhttps://doi.org/10.1097/01.mph.0000153442.42030.74Google Scholar
- Herr W, Plachter B. Cytomegalovirus and varicella-zoster virus vaccines in hematopoietic stem cell transplantation. Expert Rev Vaccines. 2009; 8(8):999-1021. PubMedhttps://doi.org/10.1586/erv.09.58Google Scholar
- Hata A, Asanuma H, Rinki M, Sharp M, Wong RM, Blume K, Arvin AM. Use of an inactivated varicella vaccine in recipients of hematopoietic-cell transplants. N Engl J Med. 2002; 347(1):26-34. PubMedhttps://doi.org/10.1056/NEJMoa013441Google Scholar
- Redman RL, Nader S, Zerboni L, Liu C, Wong RM, Brown BW, Arvin AM. Early reconstitution of immunity and decreased severity of herpes zoster in bone marrow transplant recipients immunized with inactivated varicella vaccine. J Infect Dis. 1997; 176(3):578-85. PubMedhttps://doi.org/10.1086/514077Google Scholar
- Meyers JD, Flournoy N, Thomas ED. Cell-mediated immunity to varicella-zoster virus after allogeneic marrow transplant. J Infect Dis. 1980; 141(4):479-87. PubMedhttps://doi.org/10.1093/infdis/141.4.479Google Scholar
- Kato S, Yabe H, Yabe M, Kimura M, Ito M, Tsuchida F. Studies on transfer of varicella-zoster-virus specific T-cell immunity from bone marrow donor to recipient. Blood. 1990; 75(3):806-9. PubMedGoogle Scholar
- Distler E, Schnurer E, Wagner E, von Auer C, Plachter B, Wehler D. Recovery of varicella-zoster virus-specific T cell immunity after T cell-depleted allogeneic transplantation requires symptomatic virus reactivation. Biol Blood Marrow Transplant. 2008; 14(12):1417-24. PubMedhttps://doi.org/10.1016/j.bbmt.2008.09.004Google Scholar
- Thomas S, Herr W. Natural and adoptive T-cell immunity against herpes family viruses after allogeneic hematopoietic stem cell transplantation. Immunotherapy. 2011; 3(6):771-88. PubMedhttps://doi.org/10.2217/imt.11.47Google Scholar
- Arvin AM. Humoral and cellular immunity to varicella-zoster virus: an overview. J Infect Dis. 2008; 197(Suppl 2):S58-60. PubMedhttps://doi.org/10.1086/522123Google Scholar
- Arvin AM. Cell-mediated immunity to varicella-zoster virus. J Infect Dis. 1992; 166(Suppl 1):S35-41. PubMedhttps://doi.org/10.1093/infdis/166.Supplement_1.S35Google Scholar
- Weinberg A, Levin MJ. VZV T-cell mediated immunity. Curr Top Microbiol Immunol. 2010; 342:341-57. PubMedhttps://doi.org/10.1007/82_2010_31Google Scholar
- Huang Z, Vafai A, Lee J, Mahalingam R, Hayward AR. Specific lysis of targets expressing varicella-zoster virus gpI or gpIV by CD4+ human T-cell clones. J Virol. 1992; 66(5):2664-9. PubMedGoogle Scholar
- Arvin AM, Sharp M, Smith S, Koropchak CM, Diaz PS, Kinchington P. Equivalent recognition of a varicella-zoster virus immediate early protein (IE62) and glycoprotein I by cytotoxic T lymphocytes of either CD4+ or CD8+ phenotype. J Immunol. 1991; 146(1):257-64. PubMedGoogle Scholar
- Sadzot-Delvaux C, Arvin AM, Rentier B. Varicella-zoster virus IE63, a virion component expressed during latency and acute infection, elicits humoral and cellular immunity. J Infect Dis. 1998; 178(Suppl 1):S43-47. PubMedhttps://doi.org/10.1086/514259Google Scholar
- Herr W, Ranieri E, Olson W, Zarour H, Gesualdo L, Storkus WJ. Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses. Blood. 2000; 96(5):1857-64. PubMedGoogle Scholar
- Barnstable CJ, Bodmer WF, Brown G, Galfre G, Milstein C, Williams AF, Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell. 1978; 14(1):9-20. PubMedhttps://doi.org/10.1016/0092-8674(78)90296-9Google Scholar
- Lampson LA, Levy R. Two populations of Ia-like molecules on a human B cell line. J Immunol. 1980; 125(1):293-9. PubMedGoogle Scholar
- Spits H, Borst J, Giphart M, Coligan J, Terhorst C, De Vries JE. HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes. Eur J Immunol. 1984; 14(4):299-304. PubMedhttps://doi.org/10.1002/eji.1830140404Google Scholar
- Watson AJ, DeMars R, Trowbridge IS, Bach FH. Detection of a novel human class II HLA antigen. Nature. 1983; 304(5924):358-61. PubMedhttps://doi.org/10.1038/304358a0Google Scholar
- Kramer-Albers EM, Bretz N, Tenzer S, Winterstein C, Mobius W, Berger H. Oligodendrocytes secrete exosomes containing major myelin and stress-protective proteins: Trophic support for axons?. Proteomics Clin Appl. 2007; 1(11):1446-61. PubMedhttps://doi.org/10.1002/prca.200700522Google Scholar
- Weinzierl AO, Rudolf D, Hillen N, Tenzer S, van Endert P, Schild H. Features of TAP-independent MHC class I ligands revealed by quantitative mass spectrometry. Eur J Immunol. 2008; 38(6):1503-10. PubMedhttps://doi.org/10.1002/eji.200838136Google Scholar
- Kreiter S, Konrad T, Sester M, Huber C, Tureci O, Sahin U. Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA. Cancer Immunol Immunother. 2007; 56(10):1577-87. PubMedhttps://doi.org/10.1007/s00262-007-0302-7Google Scholar
- Sharp M, Terada K, Wilson A, Nader S, Kinchington PE, Ruyechan WT. Kinetics and viral protein specificity of the cytotoxic T lymphocyte response in healthy adults immunized with live attenuated varicella vaccine. J Infect Dis. 1992; 165(5):852-8. PubMedhttps://doi.org/10.1093/infdis/165.5.852Google Scholar
- Malavige GN, Jones L, Black AP, Ogg GS. Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors. Clin Exp Immunol. 2008; 152(3):522-31. PubMedhttps://doi.org/10.1111/j.1365-2249.2008.03633.xGoogle Scholar
- Hayward AR. In vitro measurement of human T cell responses to varicella zoster virus antigen. Arch Virol Suppl. 2001; 17:143-9. PubMedGoogle Scholar
- Diaz PS, Smith S, Hunter E, Arvin AM. T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine. J Immunol. 1989; 142(2):636-41. PubMedGoogle Scholar
- Levin MJ, Oxman MN, Zhang JH, Johnson GR, Stanley H, Hayward AR. Varicella-zoster virus-specific immune responses in elderly recipients of a herpes zoster vaccine. J Infect Dis. 2008; 197(6):825-35. PubMedhttps://doi.org/10.1086/528696Google Scholar
- Kimura H, Wang Y, Pesnicak L, Cohen JI, Hooks JJ, Straus SE, Williams RK. Recombinant varicella-zoster virus glyco-proteins E and I: immunologic responses and clearance of virus in a guinea pig model of chronic uveitis. J Infect Dis. 1998; 178(2):310-7. PubMedhttps://doi.org/10.1086/515638Google Scholar
- Hasan UA, Harper DR, Wren BW, Morrow WJ. Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E. Vaccine. 2002; 20(9–10):1308-15. PubMedhttps://doi.org/10.1016/S0264-410X(01)00475-3Google Scholar
- Leroux-Roels G, Vassilev VP, Heineman T.Paper presented at: ; 2010. Google Scholar