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Tumour Immunology Laboratory, Epstein-Barr Virus Unit, Bancroft Centre, Queensland Institute of Medical Research, University of Queensland, Joint Oncology Program, Herston, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the last few years, most of the vaccine development efforts have concentrated on the use of a subunit preparation of gp350 (recombinant and affinity-purified) and have been directed toward blocking virus attachment to the target cell in the oropharynx 2 . The general approach has been to immunize cotton-top marmosets with gp350 and determine their ability to restrict the outgrowth of EBV-positive lymphomas in these animals. Indeed, highly purified gp350, when administered s.c. in conjunction with adjuvants (muramyl dipeptide or immunostimulatory complexes), induced high levels of serum-neutralizing Abs and inhibited tumor formation in cotton-top tamarins 3 . A number of recombinant vectors including vaccinia-gp350 and adenovirus 5-gp350 have also been successfully used in these animals to block tumor outgrowth 4 . The precise mechanism by which gp350 affords protection from lymphomas in cotton-top tamarins remains unclear. The fact that development of neutralizing Ab titers in vaccinated animals does not always correlate with protection indicates that gp350-specific T cell-mediated immune responses may also have an effector role 5 . Furthermore, Yao and colleagues 6 showed that very low levels of neutralizing anti-gp350 Abs are present in the saliva of healthy EBV-immune donors, which suggests that such Abs are unlikely to be the basis of long-term immunity in healthy seropositive individuals.
In the present study, we have addressed the question of whether EBV structural Ags stimulate specific T cell responses, using a novel method 7 designed to identify CTL epitopes within gp350 and gp85. Using this protocol, we firstly identified a number of HLA A2-binding peptides from gp350 and gp85. Strong ex vivo cytotoxic T cell activity against four of these peptides was noted from PBLs from infectious mononucleosis (IM)3 patients. Moreover, these effectors also showed efficient recognition of target cells infected with recombinant vaccinia-encoding gp350 and gp85. These individuals maintained gp350- and gp85-specific memory CTL, albeit at low levels, following recovery from acute IM. We also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes afforded significant protection against recombinant vaccinia virus infection, which correlated with the activation of gp350- and gp85-specific CTL in these animals. These results provide evidence that CTL epitopes from the EBV structural proteins could be exploited in the development of an EBV vaccine that confers a protective immunity against EBV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IM patients, identified on clinical grounds and by heterophile Ab positivity, were bled during the first 5–10 days of illness and, in two cases, on a second occasion 24–36 mo after the resolution of symptoms. These patients were HLA typed for the HLA A2 allele by serotyping in microcytotoxicity and by genotyping. Three patients (SB, LP, and MG) were identified as HLA A2-positive patients, and this was subsequently confirmed by FACS analysis using an HLA A2-specific mAb (American Type Culture Collection, Manassas, VA).
Establishment and maintenance of cell lines
EBV-transformed lymphoblastoid cell lines (LCLs) were established from a panel of IM and healthy EBV-seropositive donors by exogenous virus transformation of peripheral B cells using type 1 (B95.8) or type 2 (Ag876) EBV isolates 8 and were routinely maintained in RPMI 1640 containing 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin plus 10% FCS (growth medium). In addition, the peptide transporter-negative B x T hybrid cell line 174 x CEM.T2 (referred to as T2) 9 were used for peptide stabilization assays.
To generate PHA blasts, PBMC were stimulated with PHA (1 µg/ml) (Commonwealth Serum Laboratories, Melbourne, Australia), and, after 3 days, growth medium containing MLA-144 supernatant and rIL-2 (10 U/ml) was added 10 . PHA blasts were propagated with biweekly replacement of IL-2 (10U/ml) and MLA supernatant (no further PHA added) for up to 6 wk.
Establishment and preparation of CTL effectors
Acute IM PBMC effectors for use in ex vivo cytotoxicity assays were resuspended in growth medium supplemented with rIL-2 and used directly in a cytotoxicity assay (see below). To generate polyclonal CTL, 2 x 106 PBMCs from HLA A2-positive donors were cocultivated for 7 days with irradiated (8000 rad) T2 cells (responder:stimulator ratio of 25:1) presensitized with synthetic peptides (1 µg/ml) 11 . On days 7 and 14, these cultures were restimulated with peptide-sensitized T2 cells. After 18 days of culture in growth medium, the cells were used as polyclonal effectors in a standard 51Cr-release assay against peptide-sensitized autologous PHA blasts.
Synthesis of peptides
Peptides, synthesized by the Merrifield solid phase method, were purchased from Chiron Mimotopes (Melbourne, Australia), dissolved in dimethyl sulfoxide, and diluted in serum-free RPMI 1640 medium for use in standard CTL assays. The purity of these peptides was tested by mass spectrometery and showed >90% purity.
MHC stabilization assays
HLA A2-binding peptides within the gp85 and gp350 Ags were identified using a protocol described elsewhere 7 . These predicted peptides were then used in a standard MHC stabilization assay using T2 cells. Briefly, T2 cells (2 x 105) were incubated with 200 µl of each of the peptides (200 µg/ml) at 26°C for 14–16 h in serum-free medium (AIM-V; Life Technologies, Gaithersburg, MD), followed by incubation at 37°C for 2–3 h. After the incubations, HLA A2 expression was measured by FACS using a monoclonal HLA A2-specific Ab (MA2.1; American Type Culture Collection).
Vaccinia virus recombinants
Recombinant vaccinia constructs encoding the EBV structural Ags gp350 (Vacc.gp350) and gp85 (Vacc.gp85), nuclear Ag 2 (EBNA2) and a vaccinia virus construct made by insertion of the pSC11 vector alone and negative for thymidine kinase (Vacc.TK-) have been previously described 12 . Target cells were infected with recombinant vaccinia virus at a multiplicity of infection of 10:1 for 1 h at 37°C, as described earlier 13, 14 . After overnight infection, cells were washed with growth medium and processed for CTL assays or for immunoblotting to assess the expression of recombinant EBV Ags 15 .
Cytotoxicity assay
Target cells were either infected with recombinant vaccinia viruses or presensitized with synthetic peptide epitopes and then incubated with 51Cr for 90 min. Following incubation, these cells were washed in growth medium and used as targets in a standard 5-h 51Cr-release assay 8 .
Limiting dilution analysis (LDA)
PBMC from post-IM donors were distributed in graded numbers
(twofold dilutions) from 6.25 x 103 to 5 x
104 cells per well in round-bottom microtiter plates.
Approximately 5 x 104 
-irradiated (2000 rad)
peptide-sensitized (1 µg/ml) autologous PBMC were added to give a
total volume of 100 µl. Twenty-four replicates were used at each
concentration in each experiment. Cultures were fed on days 4 and 7
with 50 µl of medium supplemented with 20 U of rIL-2 and 30% (v/v)
supernatant from MLA-144 cultures. On day 10, each CTL microculture was
split into two replicates and used as effectors in a standard 5-h
51Cr-release assay against autologous PHA blasts precoated
with structural (gp350 or gp85) or latent (EBNA3 or LMP2A) Ag peptide
epitopes or left uncoated. Wells were scored as positive when the
percent specific 51Cr release for peptide-sensitized target
cells exceeded the mean release from untreated control wells by 3 SD.
LDA was performed by the method of maximum likelihood estimation 7 .
Data from all experiments were compatible with the hypothesis of
single-hit kinetics (p > 0.4), and precursor
estimates are given with 95% confidence limits.
Immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes
HLA A2/Kb transgenic mice used in this study have
been described elsewhere 16 (a kind gift from Dr. L. Sherman, Scripps
Research Institute). These mice express a chimeric class I molecule
composed of the 
1 and 2 domains of the human A*0201 allele and
the 3 domains of the mouse H-2Kb class I molecules.
Peptide immunizations were conducted as described by Vitiello
and colleagues 17 . Briefly, these animals were twice immunized (at a
14-day interval) s.c. with 50 µg/mouse of CTL epitopes emulsified in
IFA together with 5 µg tetanus toxoid as a source of help. Four weeks
following peptide immunization, animals were assessed for gp350- and
gp85-specific CTL response. For assessing these CTL responses,
splenocytes (3 x 106 cells/ml) were cocultured with
syngeneic, irradiated (2000 rad) peptide-coated LPS blasts (3 x
105 cells/ml) and 3 µg/ml human
ß2-microglobulin. CTL activity was tested on day 6 using
a standard 51Cr-release assay.
Vaccinia protection assay
For protection experiments, groups of 8-wk-old female A2/Kb transgenic mice were immunized with CTL epitopes as described above. On day 28, mice were challenged with Vacc.gp85 and Vacc.gp350 i.p. (1 x 107 plaque-forming units in 100 µl PBS). After 4 days of challenge, these animals were sacrificed and vaccinia titers measured in both ovaries by plaque assay on confluent CV1 cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify potential HLA A2-restricted epitopes within gp85 and
gp350, the amino acid sequence was analyzed by a computer program
designed to predict HLA-binding peptides, based on an estimation of the
half-time disassociation of the HLA-peptide complex
(http://bimas.dcrt.nih.gov/molbio/hla bind/index.html) 18 . A total
of 20 peptides (13 from gp85 and 7 from gp350) with an estimated
half-time disassociation score of >100 for gp85 and >50 for gp350
were selected (Table I
). These peptides
were then tested for HLA A2-binding efficiency using HLA A2-positive T2
cells. Representative data from a series of experiments is presented in
Fig. 1. This analysis showed that seven
of these peptides significantly increased the expression of HLA A2 on
T2 cells, suggesting that these peptides might be potential HLA
A2-restricted epitopes.
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The seven HLA A2-binding peptides, which included four peptides
from gp85 and three peptides from gp350, were next tested for CTL
recognition by effectors from IM patients. In addition, we also
included an HLA A2-restricted CTL epitope from EBV latent membrane
protein 1 (LMP1) as a positive control 11 . PBMCs from three HLA
A2-positive IM patients, SB, LP, and MG, were resuspended in
IL-2-supplemented growth medium and used as effectors in a standard
51Cr-release assay against HLA-matched PHA blasts
sensitized with the gp85, gp350, or LMP1 peptides. Representative data
from two different experiments is shown in Fig. 2, A–C. Effectors from all
three IM patients showed clear recognition of the reference LMP1
peptide (YLQQNWWTL), consistent with our earlier finding that this
peptide is recognized by EBV-specific CTL. More importantly, these IM
patients also showed strong ex vivo recognition of target cells
sensitized with selected gp85 and gp350 peptides, and the level of CTL
recognition of target cells sensitized with these peptides was
comparable with that seen with the LMP1 peptide (YLQQNWWTL).
Interestingly, each of these individuals showed a distinct pattern of
reactivity against these peptides. IM patient SB showed strong
reactivity against peptides SLVIVTTFV (gp85) and VLQWASLAV (gp350)
(Fig. 2A), while the LP and MG effectors recognized target
cells preloaded with peptides LMIIPLINV (gp85) and VLQWASLAV
(gp350) (Fig. 2, B and C). Furthermore, ex vivo
effectors from patient LP also recognized target cells infected with
Vacc.gp350 and Vacc.gp85 (Fig. 2D).
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The data presented above clearly demonstrate that gp85 and gp350
include CTL determinants that can bind HLA A2 molecules and are
efficiently recognized by ex vivo effectors from IM patients. To
determine whether gp85- or gp350-reactive CTL can be detected following
recovery from IM, PBMCs from the two donors, SB and LP, were collected
at 24–36 mo post-IM, respectively, and were stimulated with T2 cells
presensitized with each of the gp85 and gp350 peptides, which showed
strong HLA A2 binding. On day 18, these CTL effectors were tested
against peptide-sensitized autologous PHA blasts. Representative data
from polyclonal CTL from donor SB are presented in Fig. 3. CTL effectors from donor SB not only
showed strong reactivity against peptides SLVIVTTFV and VLQWASLAV
but also recognized two other peptides from gp85 (LMIIPLINV and
TLFIGSHVV). Donor LP also showed a similar pattern of CTL lysis. Thus
peptide TLFIGSHVV was a target for EBV-specific CTL recognition in the
memory response of these A2-positive individuals, but this response was
not detectable with ex vivo effectors during acute infection. Another
important point that needs to be highlighted here is that our attempts
to activate gp85- or gp350-specific CTL with autologus LCLs as
stimulators were unsuccessful. This result is not surprising because it
is well established that in latently infected B cells, gp350 or gp85
Ags are poorly expressed. The LCL-stimulated polyclonal T cell lines
from these donors are strongly reactive against latent Ags (data
not shown). This observation is consistent with our earlier studies,
which showed that CTL responses in healthy virus carriers is often
dominated by reactivity to latent Ags 13 .
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Having established that gp85 and gp350 includes CTL epitopes, we
extended our studies to explore the possibility of using these peptide
epitopes to induce specific CTL response in vivo. HLA A2/Kb
transgenic mice were used as an experimental model to address this
issue. These mice express a chimeric class I molecule composed of the
1 and 2 domains of the human A*0201 allele and the 3 domain of
the mouse H-2Kb class I molecules. These animals were
immunized s.c. with gp350 or gp85 CTL epitopes emulsified in IFA
together with tetanus toxoid as a source of help. The SLVIVTTFV and
TLFIGSHVV peptides from gp85 and the VLQWASLAV peptide from gp350 were
used for immunization. Two weeks following immunization, specific CTL
response was assessed in each mouse using splenocytes or pooled
inguinal lymph node cells as effectors. Data presented in Fig. 5, A–C demonstrate that
peptide epitopes from gp85 (SLVIVTTFV and TLFIGSHVV) and gp350
(VLQWASLAV) induced strong CTL response in splenocytes. Interestingly,
CTL activated from splenocytes with peptide TLFIGSHVV consistently
showed strong lysis of targets from all the animals tested, while
splenocytes from SLVIVTTFV- and VLQWASLAV-immunized mice showed
variable in vitro CTL lysis. In two different sets of experiments, only
50–60% of the animals immunized with SLVIVTTFV and
VLQWASLAV showed strong CTL activity in splenocytes. On the other
hand, a strong specific CTL activity was consistently observed in
pooled lymphocytes from inguinal lymph nodes (Fig. 5D).
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Four weeks after peptide immunization with gp85 or gp350 CTL
epitopes, HLA A2/Kb mice were challenged with
107 plaque-forming units of recombinant vaccinia virus
encoding either gp85 or gp350. After 4 days of challenge, these animals
were sacrificed and vaccinia titers measured in both ovaries by plaque
assay on confluent CV1 cells. Data from one such experiment is
presented in Fig. 6. Animals immunized
with gp85 and gp350 epitopes showed very low to undetectable virus in
their ovaries, while in naive mice very high titers of vaccinia virus
were detected. This protection correlated with strong induction of
epitope-specific CTL responses detected in the splenocytes and lymph
node cells collected 4 wk after primary peptide vaccination in HLA
A2/Kb transgenic mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EBV structural Ags, primarily gp350, have long been considered as the potential candidates for an EBV vaccine. The suggestion that gp350 is a likely vaccine candidate was based initially upon the observation that this glycoprotein is the principal target of the virus-neutralizing Ab response 19 . A number of recombinant formulations of gp350, either presented as a subunit Ag or expressed from recombinant viral vectors, designed to induce high titer neutralizing Abs, have shown significant protection against EBV-induced B cell lymphomas in cotton-top tamarins 2 . However, development of neutralizing Abs in vaccinated animals does not always shows limited correlation with protection from EBV infection, although recent results have suggested a role for gp350-specific CTL in this protection 5 . If the latter suggestion is correct, it is important to identify the potential CTL determinants within EBV structural proteins because it is now well established that immunization with whole viral proteins is unable to elicit an efficient CTL response. Moreover, a vaccine based on CTL epitopes provides an opportunity to include determinants not only from gp350 but also from other structural Ags, such as gp85. To address this issue we have used a novel protocol to successfully identify CTL epitopes within gp350 and gp85. In the first set of experiments, we identified HLA A2-binding peptides within gp350 and gp85. Subsequent experiments were focussed on IM patients with the HLA A2 allele. Using ex vivo primary effectors, we observed strong reactivity to three different gp350 and gp85 peptides, and the levels of CTL lysis was comparable to that seen with LMP1 peptide. Interestingly, individual IM patients showed distinct patterns of reactivity to each of these peptides. Strong reactivity against peptides SLVIVTTFV (gp85) and VLQWASLAV (gp350) was observed with ex vivo effectors from patient SB, while the LP and MG effectors recognized target cells preloaded with LMIIPLINV (gp85) and VLQWASLAV (gp350) peptides. More importantly, ex vivo effectors from patient LP also recognized target cells infected with Vacc.gp350 and Vacc.gp85. Interestingly, the level of ex vivo CTL lysis directed to epitopes from structural Ags was consistently higher than those seen in the same assays against HLA A2-restricted CTL epitopes from a latent Ag. These results are consistent with recent observations by Steven and colleagues 20 that ex vivo CTL reactivity to lytic Ags in IM patients is significantly higher compared with latent Ags.
In the next set of experiments, we explored the possibility of detecting structural Ag-specific CTL responses in individuals following resolution of IM symptoms. This follow up analysis was conducted 24–36 mo after acute IM. Our initial attempts to isolate gp350- or gp85-specific CTL from post-IM donors by stimulating with the autologous LCL were unsuccessful. Subsequently, we used peptide-loaded T2 cells as stimulators to generate gp350- and gp85-specific CTL. We have recently shown that this method can be successfully used to raise low-frequency EBV-specific CTL precursors 11 . Stimulation of PBMC from donors SB and LP raised strong CTL responses to the gp85 and gp350 CTL epitopes. Both donors SB and LP not only showed reactivity against peptides SLVIVTTFV, LMIIPLINV, and VLQWASLAV but also recognized another peptide from gp85, TLFIGSHVV. It is interesting to note here that both donors showed no ex vivo CTL reactivity to TLFIGSHVV during acute IM. One of the important conclusions drawn form these analyses is that, following recovery from acute IM, there is a significant reduction in CTL precursors to the structural Ags, and the response becomes dominated by CTL reactive to the latent Ags. Indeed, LDA for CTL precursors specific for the gp350 or gp85 peptide epitopes in donors SB and LP post-IM showed frequencies of >1/50,000, while much higher precursor frequencies for CTL epitopes within EBNA proteins were seen.
The detection of a strong ex vivo CTL response in IM patients to the structural Ags has important implications for any future vaccine design. As mentioned above, to date, the major emphasis of vaccine design based on EBV structural Ags has been directed toward generating a strong neutralizing Ab response. However, these neutralizing Ab responses fail to correlate with protection against EBV-induced polyclonal lymphomas in cotton-top marmosets. Nevertheless, it is possible that this protection is mediated by structural Ag-specific CTL responses. To address this issue, we employed an experimental animal model system to determine whether gp350 or gp85 CTL epitope-immunized transgenic mice, expressing the human HLA A2 Ag, are capable of 1) generating structural Ag-specific CTL responses and 2) reducing infection with a recombinant vaccinia virus infection expressing the gp350 or gp85 Ag. These mice not only showed induction of a strong CTL response following immunization, but also acquired strong resistance to virus infection. It is important to mention here that although this experiment does not allow any firm conclusions on the efficacy of a gp350 and/or gp85 CTL epitope-based vaccine in humans, it does clearly show that CTL epitopes from the EBV structural Ags can be used as immunogens to induce an efficient CTL response in vivo. Moreover, this approach also overcomes limitations of whole gp350 or gp85 proteins that might be inefficient at eliciting CTL responses in humans. Obviously, one of the possible obstacles of any epitope-based approach to vaccination in humans is HLA polymorphism because epitope choice is allele-specific. However, this obstacle might be overcome using appropriate mixtures of synthetic peptide epitope or by constructing vectors to express polypeptides in which the relevant epitope sequences are linearly joined together. Indeed, earlier studies from our laboratory have shown that if such an EBV polyepitope sequence is expressed within cells from a recombinant vaccinia vector, all of the constituent epitopes are efficiently presented for CTL recognition 21 , indicating the potential of this approach as a vaccine strategy. More recently, work in a murine model has also shown that each of several CTL epitopes combined in a polyepitope construct was capable of eliciting a CTL response in vivo and could protect the animals from subsequent challenge 22 . In the long term, it may be possible to combine CTL epitopes from the EBV structural Ags with latent Ag epitopes generating a chimeric protein that fuses the important immunogenic determinants from the two different types of Ags to design an effective vaccine.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rajiv Khanna, Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Brisbane, Australia 4029. E-mail address:
3 Abbreviations used in this paper: LMP1, latent membrane protein 1; IM, infectious mononucleosis; LDA, limiting dilution analysis; LCL, lymphoblastoid cell line.
Received for publication August 3, 1998. Accepted for publication November 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), SLVIVTTFV (
), and TLFIGSHVV (
•) is shown. CTL activity was tested on day 6 using a standard
51Cr-release assay.