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Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-10-3092.
NEOPLASIA
From the Cooperative Research Centre for
Vaccine Technology, Tumour Immunology Laboratory, Division of
Infectious Diseases and Immunology, Queensland Institute of Medical
Research and Joint Oncology Program, Department of Molecular and
Cellular Pathology, University of Queensland, Brisbane,
Australia; Synthetic Vaccine Laboratory, John Curtin
School of Medical Research, Australian National University, Canberra,
Australia.
Development of an epitope-based vaccination strategy designed to
enhance Epstein-Barr virus (EBV)-specific CD8+
cytotoxic T lymphocytes (CTLs) is increasingly being considered as
a preferred approach for the treatment of EBV-associated relapsed Hodgkin disease (HD) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane proteins, LMP1 and LMP2, are the only target antigens available for therapeutic augmentation of CTL responses in patients with HD and NPC. Here, we describe preclinical studies using a recombinant poxvirus vaccine that encodes a polyepitope protein comprising 6 HLA A2-restricted epitopes derived from LMP1. Human cells
infected with this recombinant polyepitope construct were efficiently
recognized by LMP1-specific CTL lines from HLA A2 healthy individuals.
Furthermore, immunization of HLA A2/Kb mice with this
polyepitope vaccine consistently generated strong LMP1-specific CTL
responses to 5 of the 6 epitopes, which were readily detected by both
ex vivo and in vitro assays. More important, this polyepitope vaccine
successfully reversed the outgrowth of LMP1-expressing tumors in HLA
A2/Kb mice. These studies provide an important platform for
the development of an LMP-based polyepitope vaccine as an
immunotherapeutic tool for the treatment of EBV-associated HD and NPC.
(Blood. 2003;101:3150-3156) The concept of a role for the immune system in the
control and elimination of virus-infected malignant cells has existed
for many years, giving rise to the theory of immunologic surveillance against tumors. More recently, it has been established that tumor rejection is mediated by lymphocytes and, most notably, by cytotoxic T
lymphocytes (CTLs). This concept is based mainly on the assumption that, like normal virus-infected cells, tumor cells can present virus-specific epitopes on their surface in conjunction with major histocompatibility complex (MHC) molecules, which can be
recognized by CTLs. Indeed, Epstein-Barr virus (EBV)-associated
Hodgkin disease (HD) and nasopharyngeal carcinoma (NPC) represent 2 of
the classic examples of virus-infected malignancies that are
characterized by the expression of EBV nuclear antigen 1 (EBNA1) and
latent membrane proteins (LMP) 1 and 2.1,2 Molecular
analysis of both NPC and HD have shown that unlike other EBV-associated
malignancies, such as Burkitt lymphoma, in vitro-established
tumor cell lines from these malignancies are highly susceptible to
lysis by EBV-specific CTLs and express normal levels of
antigen-processing genes (TAP-1 and
TAP-2).3,4
In spite of the highly immunogenic phenotypes associated with these
malignancies, tumor cells in NPC and HD can escape CTL-mediated immune
control in vivo. A number of possible mechanisms have been proposed to
explain this immune evasion strategy. Immunologic and biochemical
analysis of LMP1 sequences associated with NPC and HD have shown that
these sequences are not only highly oncogenic but also seem to be
poorly immunogenic in murine models when compared with the LMP1
sequences derived from normal EBV-infected B cells.5,6 Moreover, independent studies by different groups also have shown that
Reed-Sternberg cells in HD secrete anti-inflammatory cytokines (such as
interleukin 10 [IL10] and tumor growth factor It has been proposed that strategies designed to augment LMP1- and/or
LMP2-specific CTL responses in HD and NPC patients may provide a
therapeutic benefit for these malignancies. Indeed, adoptive transfer
of polyclonal EBV-specific autologous CTLs into patients with advanced
HD recently has been reported.8 In this study, all
patients who received this adoptive therapy had incidental improvement,
including reduction of high virus load, increase in virus-specific CTL
precursor frequency, resolution of some symptoms, and stabilization of
the disease. Unfortunately, all of these patients failed to recover
from the advanced stages of HD. One of the major limitations of this
approach was that the CTLs transferred in this study were
expanded by stimulating the peripheral blood lymphocytes with
autologous EBV-transformed lymphoblastoid cell lines (LCLs), which are
known to preferentially stimulate T cells specific for EBNA antigens
rather than LMP1 and LMP2.9 In spite of these
limitations, it was encouraging to see a short-term therapeutic effect,
and further improvement of the CTL activation strategy may allow
selective expansion of T cells that are specific for a limited range of
viral antigens expressed in HD and NPC. In the present study, we have
developed a novel strategy based on an LMP1 polyepitope vaccine, which
allows an efficient activation of LMP1-specific CTL responses in vivo,
and these CTLs in turn provide a strong therapeutic benefit against
LMP1-expressing tumors.
Construction of a recombinant LMP polyepitope expression
vector
Establishment and maintenance of human and mouse cell
lines
Immunization of HLA A2/Kb transgenic mice with an LMP
polyepitope vaccine
Synthesis of peptides Peptides, synthesized by the Merrifield solid phase method, were purchased from Chiron Mimotopes (Melbourne, Australia), dissolved in dimethyl sulphoxide, and diluted in serum-free RPMI 1640 medium for use in standard CTL assays. Purity of these peptides were tested by mass spectrometry and showed more than 90% purity.Assessment of T-cell responses by IFN-  (IFN-) ELISPOT method described
previously.13,14 Ninety-six well-mixed cellulose ester
membrane plates (Millipore, Bedford, MA) were coated overnight at 4°C
with 75 µL/well of 8 µg/mL anti-IFN-, capture monoclonal
antibodies (mAbs) (BD PharMingen, Los Angeles, CA; clone
R4-6A2) in freshly prepared filter-sterilized 0.1M
NaHCO3, pH 8.4. The plates were washed 6 times with
phosphate-buffered saline (PBS) prior to blocking for 1 hour at
37°C with PBS containing 5% FCS. Splenocytes were harvested into
growth medium supplemented with 5 × 105 M
2-mercaptoethanol. These cells were plated in ELISPOT plates (106/well), and then synthetic peptide epitopes were added
into the wells at a final 1 µ/g concentration. For negative controls,
splenocytes were incubated without peptide. The plates were incubated
at 37°C with 5% CO2 overnight (about 16-20 hours), then
washed 3 times with PBST (0.05% Tween-20 in PBS) and 3 times
with PBS before addition of 75 µL/well of 1 µg/mL
anti-IFN--biotin (PharMingen, La Jolla, CA; clone XMG1.2)
and incubated at room temperature for a further 4 hours. After
incubation, plates were washed again 3 times each with PBS-Tween (PBST)
and PBS, and 100 µL/well of 1 µg/mL streptavidin-alkaline
phosphatase conjugate was added and incubated at room temperature for 2 hours. After a final PBS wash, BCIP/NBT (5-bromo, 4-chloro,
3-indoylphosphate/nitroblue tetrazolium) developing/substrate solution
(Sigma) was added at 100 µL/well and kept at room temperature until
individual IFN--producing cells were detected as dark spots (3-5 minutes). Color development was stopped by thoroughly washing the
plates in tap water prior to drying. Spots were counted automatically
using an image analysis software (ImagePro)15 and were
expressed as spot-forming cells (SFCs) per 106 PBMCs. The
number of IFN--secreting T cells was calculated by first correcting
for the background by the subtraction of the negative control SFC.
Establishment of human polyclonal CTL lines and LMP1-specific CTL clones Polyclonal CTL lines and LMP1-specific CTL clones were established according to previously published methods.3 Briefly, 2 × 106 PBMCs from 2 HLA A2-positive healthy virus carriers (referred to as donor no. 1 and donor no. 2) were stimulated with 1 × 106 autologous lymphocytes (responder-to-stimulator ratio of 2:1) pulsed with 10 µM peptide for 1 hour. After 3 days, growth medium with IL-2 (10 U/mL) was added, and the cells were further expanded. These lymphocytes were restimulated on day 7 with-irradiated (8000 rad) autologous LCLs. After 10 days in
culture medium, the cells were used as polyclonal effectors in a
standard 51Cr-release assay against peptide-sensitized
autologous phytohemagglutinin (PHA) blasts.
To generate peripheral blood CTL clones specific for the LMP1-derived peptides, PBMCs (2 × 106) of healthy donors were reactivated with peptide-sensitized (10 µg of peptide/mL) autologous lymphocytes (1 × 106) in 2-mL wells of a 24-well plate in growth medium. After 3 days, the cells were seeded onto 0.35% low melting gel agarose and maintained in T-cell growth medium containing recombinant IL2 (rIL2) (50 IU/mL). After another 3 days, growing clones were transferred to 96-well round-bottom tissue culture plates (Life Technologies, Sydney, Australia) and cultured in T-cell growth medium containing rIL2 (50-100 IU/mL). In vitro cytotoxicity assays Target cells presensitized with synthetic peptide epitopes or infected with recombinant vaccinia encoding the LMP polyepitope were incubated with 51Cr for 90 minutes. Following incubation, these cells were washed in growth medium and used as targets in standard 5-hour 51Cr-release assays.In vivo cytotoxicity assays LMP1 epitope-specific in vivo CTL activity was assessed by using carboxyfluorescein diacetate succinimidyl ester (CFSE) dye-labeled (Molecular Probes, Eugene, OR) target cells.16 Briefly, HLA A2/Kb splenocyte cell suspensions were divided into 2 populations following red cell lysis. One population was pulsed with an LMP1 epitope (1 µg/mL) for 90 minutes at 37°C, washed in PBS, and labeled with a high concentration (5 µM) of CFSE. Control, uncoated target cells were labeled with a low concentration of CFSE (0.5 µM). Cells (107) of each population were mixed in 200 µL of PBS and injected intravenously into Vacc.polyLMP- or Vacc.TK -immunized mice. Specific in vivo
cytotoxicity was determined by collecting the cells from spleen from
recipient mice 18 hours after injection, and the number of cells in
each target cell population was determined by flow cytometry. The ratio
between the percentages of uncoated versus LMP1 peptide-coated
(CFSElow/CFSEhigh) cells was calculated to
obtain a numeric value of cytotoxicity.
Tumor challenge and polyepitope immunization To assess the efficacy of the LMP polyepitope vaccine, 2 different vaccination strategies were used. In the first set of experiments, HLA A2/Kb mice were immunized intraperitoneally with either Vacc.polyLMP or Vacc.TK
(107 PFU/mouse). These mice were challenged subcutaneously
with live 107 EL4-A2/Kb-LMP1 cells 3 weeks
after the immunization. Following challenge, these animals were
regularly monitored for 21 days, and the tumor size measured by a
caliper. In the second set of experiments, HLA A2/Kb mice
were first challenged with EL4-A2/Kb-LMP1 (107
cells/mouse) tumor cells. These mice were immunized with either Vacc.polyLMP or Vacc.TK 12 days after the challenge, when
the tumor size was approximately 0.4 cm in diameter. The therapeutic
efficacy of the LMP polyepitope vaccine was assessed by regular
monitoring of tumor regression. Any mice showing a tumor size larger
than 1.0 cm in diameter were killed according to the guidelines of the
institute animal ethics committee.
LMP1-specific CTL lines efficiently recognize target cells infected with a recombinant LMP polyepitope A recombinant LMP polyepitope vaccinia virus (Vacc.polyLMP) encoding 6 different HLA A2-restricted epitopes (Table 1) was derived using homologous recombination. To test whether the LMP1 epitopes encoded by this polyepitope were endogenously processed, target cells infected with Vacc.polyLMP were exposed to LMP1-specific CTL polyclonal/clonal lines specific for YLLEMLWRL (referred to as YLL) and YLQQNWWTL (referred to as YLQ) epitopes. These CTL lines were generated from healthy virus carriers, and their specificity was confirmed by their ability to lyse HLA A2-positive target cells coated with respective peptide epitopes (Figure 2A). HLA A2-positive fibroblasts infected with Vacc.polyLMP also were efficiently recognized by YLL- and YLQ-specific CTL lines (Figure 2B). These results clearly show that HLA class I-restricted CTL epitopes included in the LMP polyepitope are efficiently processed and presented to the target cells.
Generation of LMP1-specific CTL responses in HLA A2/Kb mice vaccinated with a LMP polyepitope To determine whether the LMP polyepitope construct was capable of raising CTL responses in vivo, HLA A2/Kb transgenic mice were vaccinated with either the Vacc.polyLMP or Vacc.TK, and
21 days following immunization, CTL responses to each of the 6 epitopes
were assessed. Three different methods were used to assess T-cell
responses. In the first set of experiments, ex vivo T-cell reactivity
to each of the peptide epitopes was assessed by ELISPOT technology. A
minimum of 6 animals was assessed in each group. Splenocytes from these
mice were used as responder cells for the detection of
epitope-responsive T cells. Data presented in Figure
3 clearly show that all mice consistently
responded to 5 (YLL, YLQ, TLL, LLV, and LLL) of the 6 LMP1 CTL epitopes included in the polyepitope vaccine. On the other hand, low levels of
T-cell response to RLG epitope were detected (Figure 3).
In the next set of experiments, these precursor T cells were stimulated
in vitro to determine whether epitope-specific CTL effectors could be
expanded following immunization with the LMP polyepitope.
Representative data from a group of 6 animals are shown in Figure
4. Following a single stimulation with
peptide-sensitized LPS blasts, strong CTL responses to 2 epitopes (YLL
and TLL) were detected, while a low-to-moderate response to the LLV
epitope was detected. No epitope-specific lysis was detected for LLL
and YLQ epitopes. However, a significant increase in the levels of CTL
lysis was observed following secondary stimulation with
peptide-sensitized LPS blasts. It is important to mention here that no
CTL effectors were expanded following stimulation with the RLG peptide.
These observations are consistent with our ELISPOT data presented in Figure 3.
Finally, LMP1 epitope-specific CTL activity was observed in
Vacc.polyLMP-immunized mice using an in vivo cytotoxicity assay to
monitor depletion of target cells labeled with immunogenic peptide and
CFSE dye. Vacc.polyLMP and Vacc.TK
Immunization with Vacc.polyLMP affords protection against LMP1-expressing tumors To test whether the Vacc.polyLMP vaccine-induced T-cell responses can afford protection against LMP1-expressing tumor cells, 2 groups of HLA A2/Kb mice (10 mice in each group) were first immunized with Vacc.polyLMP or Vacc.TK and then challenged with
EL4-A2/Kb-LMP1 cells. These mice were regularly monitored
for tumor outgrowth. Although both groups of animals developed tumors,
the tumor outgrowth in Vacc.TK was highly aggressive and
showed no evidence of protection from tumor challenge (Figure
6). On the other hand, these tumors grew much less aggressively in animals immunized with Vacc.polyLMP, and this
outgrowth was completely resolved in 90% of the animals by the end of
the observation period. By day 24, the average tumor load in
Vacc.polyLMP-immunized mice was 30- to 33-fold lower when compared with
Vacc.TK-immunized mice (Figure 6). It is important to
mention here that animals immunized with Vacc.TK or
Vacc.polyLMP showed no protection against challenge with
EL4/A2Kb cells, indicating that the epitope-specific immune
response was critical for this protection (data not shown).
The use of the LMP1 polyepitope vaccine as a therapeutic tool for the
treatment of an actively proliferating tumor is one of the major
challenges for its translation for human use. To explore the
therapeutic efficacy of the polyepitope vaccine, a group of 40 HLA
A2/Kb mice were challenged subcutaneously with 1 × 107
EL4-A2/Kb-LMP1 cells and monitored for tumor load. On day 12, when the
tumor size was approximately 0.4 cm in diameter, mice were divided into
2 groups and immunized with Vacc.TK
Comparative analysis of T-cell responses to LMP1 epitopes in HLA A2/Kb mice following tumor challenge To determine whether the CTL responses in vivo correlated with the tumor protection, LMP1-specific T-cell responses were assessed following tumor challenge. Data from one such analysis is presented in Figure 8. All animals immunized with Vacc.polyLMP vaccine and showing complete protection from tumor challenge demonstrated strong ex vivo LMP1 epitope-specific CTL responses. One of the interesting aspects of this result was that a significant increase in the T-cell response to the RLGATIWQL epitope was observed following tumor challenge. Our initial studies (Figure 3) had indicated that very low levels of CTL responses are generated to RLGATIWQL epitope following immunization with Vacc.polyLMP vaccine. Furthermore, the levels of T-cell responses to the LMP1 epitopes in individual mice showed strong correlation with the tumor load on day 21. Those mice with higher precursor frequency for LMP1 epitopes showed complete resolution of tumor, whereas small tumors were evident in those mice that had lower precursor frequency. These observations clearly indicate that a strong T-cell response to LMP1 is crucial for a successful rejection of the LMP-expressing tumors. This contention is further supported by lack of tumor protection in Vacc.TK-immunized mice, which showed 2- to 2.5-fold lower
LMP1 epitope-specific T-cell responses following tumor
challenge.
Over the past several decades, the goal of curing the majority of patients with primary HD and NPC has been reached. In the past 3 decades, 2 powerful tools, irradiation and multiagent chemotherapy, have emerged as the mainstays of modern treatment.17 Although both these therapeutic strategies are most efficient in eradicating a proportion of tumors, the nonspecific nature of these treatments often results in significant side effects, including long-term toxicities, development of secondary cancers, and infectious complications.18 Moreover, a small but significant proportion of HD and NPC patients relapse following chemotherapy and radiotherapy and also fail to respond to conventional therapeutic salvage strategies. More recently, there has been an increasing emphasis on the development of novel therapeutic strategies, which are specifically designed to prime the patient's own immune system to recognize EBV antigens expressed in malignant cells of HD and NPC and to specifically destroy these cells with minimal or no associated toxicities.19,20 The present study illustrates the possibility of using multiple HLA class I-restricted LMP1 CTL epitopes as a polyepitope vaccine for the treatment of EBV-associated HD and NPC. The LMP polyepitope vaccine-induced LMP1 epitope-specific CTLs of multiple specificities in HLA-A2/Kb transgenic mice and the epitopes encoded by the recombinant virus were recognized by CTL lines from HLA A2-positive healthy seropositive donors. These observations indicate that 5 of the 6 epitopes encoded by the polyepitope construct are efficiently processed and presented by antigen-presenting cells. It is important to stress here that the polyepitope vaccine technology also overcomes the potential limitation of the use of LMP1 protein as a vaccine for the induction of antigen-specific T cells. It is now firmly established that expression of LMP1 protein alone can transform normal cells and initiate the oncogenic process. Moreover, the transmembrane localization of LMP1 restricts its accessibility to the cytosolic degradation pathways and thus limits its presentation through the classic class I pathway. Previous studies have shown that a polyepitope protein is highly unstable and is rapidly degraded through the proteasome pathway, thus allowing more efficient presentation of all the epitopes encoded within this recombinant protein. The therapeutic efficacy of the polyepitope vaccine was assessed
using a quasi-HD/NPC tumor model in HLA A2/Kb transgenic mice. This
tumor model was based on the EL4-A2/Kb cells, which express the LMP1
oncogene as a transgene. Subcutaneous injection of these tumor cells
(1 × 107 cells/mice) consistently resulted in tumor
outgrowth in more than 95% of the HLA A2/Kb mice. Prior immunization
of HLA A2/Kb mice with the LMP polyepitope vaccine provided a high
degree of protection, and the tumor outgrowth was significantly reduced when compared with the TK The data presented here provide an important platform for the future development of immunotherapeutic strategies for the treatment of EBV-associated relapsed HD and NPC. Although HLA A2 is one of most common HLA class I alleles, the wider application of LMP-based polyepitope technology will require the inclusion of CTL epitopes restricted through other HLA class I alleles prevalent in NPC endemic regions of the world (HLA A11, A24, B27, and B57). Moreover, inclusion of additional epitopes from LMP1 and LMP2 also will allow the targeting of both antigens. We anticipate that the translation of polyepitope vaccine technology for human application will require other delivery modalities that are more likely to be approved by safety and human ethics committees. These include replication deficient adenovirus, poxvirus (Modified Vaccinia Ankara) vectors, naked DNA, or transduced autologous dendritic cells.21-23 Considering the highly immunosuppressive nature of malignant cells of HD, it is possible that LMP1 and LMP2 CTL induction might need to be facilitated by codelivery of cytokines and/or prime boost strategies.24
Submitted October 15, 2002; accepted November 22, 2002.
Prepublished online as Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-10-3092.
J.D. and M.S. contributed equally to this work, and their order should be considered arbitrary.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Rajiv Khanna, Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Rd, Brisbane, Australia 4029; e-mail: rajivk{at}qimr.edu.au.
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© 2003 by The American Society of Hematology.
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  C. Gurer, T. Strowig, F. Brilot, M. Pack, C. Trumpfheller, F. Arrey, C. G. Park, R. M. Steinman, and C. Munz Targeting the nuclear antigen 1 of Epstein-Barr virus to the human endocytic receptor DEC-205 stimulates protective T-cell responses Blood, August 15, 2008; 112(4): 1231 - 1239. |
Top
Abstract
Introduction
[TGF
) or no peptide (
). Mean lysis values (±SE) for each
group of mice are presented. A minimum of 6 animals was assessed for
each epitope.
) or
Vacc.TK