of June 18, 2013.
This information is current as
Simplex Virus Glycoprotein D
Lymphocyte Epitopes Identified from Herpes
HLA-A*0201-Restricted CD8+ Cytotoxic T
Wechsler, Anthony B. Nesburn and Lbachir BenMohamed
Xiaoming Zhu, Amir Mohebbi, Søren Buus, Steven L.
Dasgupta, Ilham Bettahi, Alex Nguyen, Michelle Wu,
Aziz Alami Chentoufi, Xiuli Zhang, Kasper Lamberth, Gargi
2008; 180:426-437; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2008 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
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The Journal of Immunology
by guest on June 18, 2013
HLA-A*0201-Restricted CD8?Cytotoxic T Lymphocyte
Epitopes Identified from Herpes Simplex Virus
Aziz Alami Chentoufi,2* Xiuli Zhang,2* Kasper Lamberth,†Gargi Dasgupta,* Ilham Bettahi,*
Alex Nguyen,* Michelle Wu,* Xiaoming Zhu,* Amir Mohebbi,* Søren Buus,†
Steven L. Wechsler,* Anthony B. Nesburn,* and Lbachir BenMohamed3*‡
Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD)
contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited
to CD4?T cell epitopes, with no CD8?T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid
sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability
CD8?T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited
high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53–61, gD70–78,
and gD278–286, significantly stabilized HLA-A*0201 molecules on T2cell lines and are highly conserved among and between HSV-1
and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-
seropositive healthy individuals, the most frequent and robust CD8?T cell responses, assessed by IFN-? ELISPOT, CD107a/b
cytotoxic degranulation, and tetramer assays, were directed mainly against gD53–61, gD70–78, and gD278–286epitopes. In addition,
CD8?T cell lines generated by gD53–61, gD70–78, and gD278–286peptides recognized infected target cells expressing native gD.
Lastly, CD8?T cell responses specific to gD53–61, gD70–78, and gD278–286epitopes were induced in HLA-A*0201 transgenic mice
following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8?T cell epitopes described herein are
potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines. The Journal of Im-
munology, 2008, 180: 426–437.
disease in newborns, cold sores, genital ulcerations, eye disease,
and fatal encephalitis in adults (1–6). Recurrent ocular herpes dis-
ease (primarily HSV-1) causes herpetic stromal keratitis, an im-
munoinflammatory and immunopathological lesion of the cornea
that can lead to blindness (1, 2, 7, 8). More than 80% of adults shed
reactivated HSV in their tears many times per year, and ?450,000
people in the U.S. have a history of recurrent ocular herpes disease
(1, 9). Recurrent genital herpes disease (primarily HSV-2), termed
herpes genitalis, also has an immunoinflammatory and immuno-
pathological course that leads to the development of genital le-
erpes simplex viruses type 1 and type 2, HSV-1 and
HSV-2, are infectious pathogens that cause serious dis-
eases at every stage of life, including fatal disseminated
sions, ulcerations, and scarring (7, 8). Genital herpes affects up to
33% of adults in the United States and the European Union (1, 7,
10). Together, ocular and genital herpes represent an enormous
reservoir for horizontal and vertical transmission (1, 11). The rapid
spread of herpes infection, which occurs mostly during unrecog-
nized asymptomatic shedding, is reflected in more than 1 million
new cases per year in the United States alone (1, 11, 12).
Despite the availability of many interventional strategies such as
sexual behavior education, barrier methods, and costly guanine
nucleoside antiviral drug therapies (e.g., Acyclovir and deriva-
tives), controlling the spread of ocular and genital herpesvirus re-
mains a challenge as clinical manifestation rates have continued to
rise over the last decade (8, 9, 13). An effective immunoprophy-
lactic and/or immunotherapeutic vaccine would be the most cost-
effective approach that could be helpful not only in developed
nations but also in underdeveloped regions such as some sub-Sa-
haran African areas, where 70% of high-risk, HIV-negative indi-
viduals and 85% of HIV-infected individuals are seropositive for
herpes (12, 14, 15). To date, however, no clinical herpes vaccine
is yet available. Several challenges face its development, including
the identification of target Ags and derived human epitopes and
uncertainty about the effector mechanisms mediating protective
Among some 11 herpes glycoproteins (2, 7, 8), the glycoprotein
D (gD)4has been successful in eliciting T cell-mediated immunity
*Laboratory of Cellular and Molecular Immunology, Eye Institute, University of
California Irvine, School of Medicine, Irvine, CA 92697;†Institute for Medical Mi-
crobiology and Immunology, Panum Institute, Copenhagen, Denmark; and‡Center
for Immunology, University of California Irvine, Irvine, CA 92697
Received for publication July 9, 2007. Accepted for publication October 4, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Public Health Service Research Grants EY14900,
EY14017, EY013191, and EY16663 from the National Institutes of Health, by the
Discovery Eye Foundation, and an by unrestricted grant from Research to Prevent
Blindness. L.B.M. is a Research to Prevent Blindness Special Award Investigator.
S.L.W. is a Research to Prevent Blindness Senior Scientific Investigator.
2A.A.C. and X.Z. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Lbachir BenMohamed, Labo-
ratory of Cellular and Molecular Immunology, University of California Irvine, Col-
lege of Medicine, Building 55, Room 202, Orange, CA 92868. E-mail address:
4Abbreviations used in this paper: gD, glycoprotein D; LCL, lymphoblastoid cell
line; MFI, mean fluorescence intensity; rVV, recombinant vaccinia virus; SFU, spot-
forming unit; Tg, transgenic; VVgD, vaccinia virus expressing gD.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 18, 2013
in mouse, rabbit, and guinea pig herpes models when delivered
emulsified in experimental Freud’s or derivative adjuvants (8, 16).
However, in the two most recent phase II/III clinical trials, the
delivery of gD with the clinical adjuvants MF59 and alum-mono-
phosphoryl lipid A only induced moderate and transient protection
or only protected previously uninfected women while failing to
protect men or HSV-1-seropositive women (4, 10, 17, 18). In both
trials, the HSV-specific Ab responses were similar to those of nat-
ural HSV infection. However, in vitro studies of T cell responses
induced by the gD vaccines were limited to IFN-? production, as
no functional human CD8?T cell gD epitopes have been previ-
ously identified (4, 10). The mapping of human minimal CD8?T
cell epitopes on gD would contribute to a better understanding of
the immune correlates of protection and would help in the devel-
opment of effective immunotherapeutic and immunoprophylactic
In the present study, we screened the amino acid sequences of
HSV-1 gD for potential human CD8?T cell epitopes by using
several computational algorithms to predict potential HLA-
A*0201 binding regions. The HLA-A*0201 supertype family is
represented in ?50% of the world’s population irrespective of
gender and ethnicity (19). Using several immunological parame-
ters, including peptide binding affinity to purified HLA-A*0201
molecules, stabilization of HLA-A*0201 molecules on T2cells by
high-affinity gD peptides, ex vivo tetramer staining, and in vitro
functional IFN-? ELISPOT and cytotoxic CD8?T cell assays, we
found three CD8?T cell epitopes: gD53–61, gD70–78, and gD278–286.
These minimal T cell epitopes are highly conserved among and
between HSV-1 and HSV-2 strains. They recalled functional HSV-
specific CD8?T cells in HLA-A*0201-positive individuals that
were seropositive for HSV-1, HSV-2, or both and in humanized
HLA-A*0201 transgenic (Tg) mice ocularly or intravaginally in-
fected with either HSV-1 or HSV-2.
Materials and Methods
HSV-1 gD open reading frames used in this study were from strain 17
(GenBank/GenPept accession no. Q69091). The 394-aa sequence of the
immature gD protein, including the 25-aa leader sequence, was screened
for HLA-A2.1-restricted epitopes using different computational algorithms
as previously described: BioInformatics and Molecular Analysis Section
(BISMAS) software (National Institutes of Health, Bethesda, MD; http://
bimas.dcrt.nih.gov/molbio/hla_bind/) and the SYFPEITHI algorithm
(http://www.syfpeithi.de/) (20). Potential cleavage sites for human protea-
some were identified using NetChop 3.0 (http://www.cbs.dtu.dk/services/
NetChop/) (21) and MHC Pathway (http://www.mhc-pathway.net) and
were also used in this screening (22).
To determine potential vaccine candidate, 10 HLA-A*0201 binding pep-
tides of herpes simplex virus type 1 (strain 17) glycoprotein D (GenBank/
GenPept accession no. Q69091) have been selected based on predictive
computational algorithms using a computer-based program accessed
through the National Institutes of Health BioInformatics and Molecular
Analysis Section (BIMAS) HLA peptide binding predictions web site
(http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) (23). Ten peptides
with high estimated half-time of dissociation (t1/2) were synthesized by
Magenex on a 9050 PepSynthesizer instrument (Milligen/Millipore) by
using solid-phase peptide synthesis and standard 9-fluorenylmethoxy car-
bonyl technology (Applied Biosystems). Peptides were cleaved from the
resin by using trifluoroacetic acid-anisole-thioanisole-anisole-ethanedithiol
(EDT)-water (87.5:2.5:2.5:2.5:5%) followed by ether (methyl-t-butyl
ether) extraction and lyophilization, as previously described (1, 11, 24–29).
The purity of peptides was between 75 and 96% as determined by reversed-
phase HPLC (Vydac C18) and mass spectroscopy (Voyager MALDI-TOF
system; Applied Biosystems). Stock solutions were made at 1 mg/ml in
PBS. All peptides were aliquoted and stored at ?20°C until assayed.
MHC-peptide binding assays
gD peptide epitope binding with purified HLA-A*0201 molecules was per-
formed as previously described (30–32). Briefly, denatured and purified
recombinant HLA-A*0201 heavy chains were diluted into a renaturation
buffer containing ?2-microglobulin and graded concentrations of the test
peptide and incubated at 18°C for 48 h, allowing equilibrium to be reached.
The concentration of peptide-HLA complexes generated was measured in
a quantitative ELISA and plotted against the concentration of peptide of-
fered (30). Because the effective concentration of HLA-A*0201 (2 nM)
used in this assay is below the equilibrium dissociation constant (Kd) of
most high-affinity peptide-HLA interactions, the peptide concentration
leading to half-saturation of the HLA is a reasonable approximation of the
affinity of the interaction.
Stabilization of HLA-A*0201 on class I-HLA-transfected B ? T
hybrid cell lines (T2lines)
To determine whether synthetic peptides could stabilize HLA-A*0201
molecule expression on the T2cell surface, peptide-inducing HLA-A*0201
up-regulation on T2cells was examined according to a protocol described
previously (33, 34). T2cells (3 ? 105/well) were incubated with different
concentration of individual gD peptide (as indicated in Fig. 1) in 48-well
plates for 18 h at 26°C. Cells were then incubated at 37°C for 3 h in the
presence of 0.7 ?l/ml Golgi Stop (BD Pharmingen) to block cell surface
expression of newly synthesized HLA-A*0201 molecules and human ?-2
microglobulin (1 ?g/ml). The cells were washed with FACS buffer (1%
BSA and 0.1% sodium azide in PBS) and stained with anti-HLA-A2.1-
specific mAb BB7.2 (BD Pharmingen) at 4°C for 30 min. After incubation,
the cells were washed with FACS buffer, fixed with 1% paraformaldehyde
in PBS, and analyzed by flow cytometry using a FACSCalibur device (BD
Biosciences). The acquired data were analyzed with CellQuest software
(BD Biosciences). Expression was measured by a FACSCalibur cytometer
(BD Biosciences) and mean fluorescence intensity (MFI) was recorded.
The percentage of MFI increase was calculated as follows: percentage MFI
increase ? (MFI with the given peptide ? MFI without peptide)/(MFI
without peptide) ? 100. Each experiment was performed three times and
means ? SD values were calculated.
HLA-A*0201 Tg mice
HLA-A*0201/Kb(referred to throughout as HLA-A*0201) Tg mice pro-
vided by Dr. F. Lemonnier (Pasteur Institute, Paris, France) and supplied
by Dr. D. Diamond (City of Hope, Duarte, CA) were bred at the University
of California, Irvine, CA. These mice represent the F1generation resulting
from a cross between HLA-A*0201/KbTg mice (expressing a chimeric
gene consisting of the ?1and ?2domains of HLA-A*0201 and the ?3
domain of H-2Kb) created on the C57BL/6 background with BALB/c mice
(The Jackson Laboratory). All studies were conducted in facilities ap-
proved by the Association for Assessment and Accreditation of Laboratory
Animal Care and according to Institutional Animal Care and Use Com-
mittee-approved animal protocols.
JA2.1 cells are human Jurkat cells that express the HLA-A*0201/Kbchi-
meric gene (25, 26), and LPS blasts were prepared as previously described
(27–29, 35). T2and RS cells (both from American Type Culture Collec-
tion) were grown as directed by the supplier.
Healthy individuals (negative for HIV and hepatitis B virus and without
any HSV infection history) were recruited at University of California Ir-
vine General Clinical Research Center. Approximately 175 ml of an indi-
vidual’s blood was drawn into yellow-top Vacutainer tubes (BD Bio-
sciences). The serum was isolated and underwent centrifugation for 10 min
at 800 ? g. The PBMCs were isolated by gradient centrifugation using
leukocyte separation medium (Cellgro). The cells were washed in PBS and
resuspended in complete culture medium consisting of RPMI 1640 me-
dium containing 10 FBS (Bio-Products) supplemented with 1? penicillin/
L-glutamine/streptomycin, 1? sodium pyruvate, 1? nonessential amino
acids, and 50 ?M 2-ME (Invitrogen Life Technologies). Aliquots of
freshly isolated PBMCs were also cryopreserved in 90% FCS and 10%
DMSO in liquid nitrogen for future testing.
HSV-1 and HSV-2 type specific serotyping
The sera collected from 61 patients were tested for HSV-1 and HSV-2
status using HerpeSelect IgG1 and HerpeSelect IgG2 HSV ELISA kits
(Focus Diagnostics) as previously described (36). Although in general this
427 The Journal of Immunology
by guest on June 18, 2013
assay gives a clear-cut result, in some instances the stereotyping was
backed up by Western blotting as previously described (37).
The HLA-A2 status was confirmed by PBMC staining with 2 ?l of an
anti-HLA-A2 mAb, BB7.2 (BD Pharmingen), at 4°C for 30 min. The cells
were washed and analyzed by flow cytometry using a FACScan (BD Bio-
sciences). The acquired data were analyzed with CellQuest software (BD
gD peptide/tetramer complex staining
The gD peptide/tetramer complexes were purchased from ProImmune.
Fresh PBMCs were analyzed for the frequency of CD8?T cells recogniz-
ing the gD peptide/tetramer complexes as previously described (1, 2, 7, 8).
The cells were incubated with gD peptide/tetramer complex for 30–45 min
at 37°C. The cell preparations were then washed with FACS buffer and
stained with PE-conjugated anti-human CD8 Ab (BD Pharmingen). The
cells were washed and fixed with 1% formaldehyde in PBS and analyzed
by flow cytometry using a FACScan (BD Biosciences). The acquired data
were analyzed with CellQuest software (BD Biosciences).
Lymphoblastoid cell lines (LCLs) were generated as we (26, 29, 38) and
others have previously described (39). Briefly, LCLs were derived by stim-
ulating PBMCs with PHA (4 ?g/ml) in RPMI 1640 plus 10% FCS for 3
days. Half of the medium was then replaced by fresh medium containing
IL-2 (10 ng/ml) and IL-7 (20 ng/ml). On day 6, the LCLs were infected
overnight with an empty vaccinia virus (control), a vaccinia virus express-
ing gD (VVgD), HSV-1, or HSV-2, each at a multiplicity of infection of
10. The next day, infected cells were washed three times and treated with
mitomycin C (50 ?g/ml). The LCLs were washed another three times
before a 6-h incubation with effector CD8?T cells at the indicated E:T
ratio. The cytotoxic activity was detected in a CD107a/b degranulation
assay as previously described (40).
In vitro generation of gD-peptide specific CD8 T cell line
CD8?T cells were isolated from PBMCs using a CD8 T cell isolation kit
II (MACS; Miltenyi Biotec). A CTL line was established by stimulating
CD8?T cells (2 ? 106/ml) with mitomycin C-treated (50 ?g/ml) autolo-
gous PBMCs (106/ml) incubated 3 h with individual peptides (20 ?M).
IL-2 (5 ng/ml; R & D Systems) was added to the culture every other day
starting on day 3. On day 7, cells were restimulated with mitomycin C-
treated autologous individual peptide-pulsed PBMCs (106/ml) and IL-7 (20
ng/ml) added every other day. On day 14, cultures were expanded and
restimulated as described above for another week and used as effector cells
for CD107 assay.
IFN-? ELISPOT assays
T cell stimulation was measured by IFN-? production in peptide-stimulated
PBMCs using a BD IFN-? ELISPOT kit (BD Pharmingen) as previously
described (41). Briefly, 5 ? 106PBMCs were stimulated with individual
gD peptides (20 ?M) or with heat-inactivated HSV-1 (strain McKrae)
(multiplicity of infection of 5) for 6 days. Then, activated PBMCs were
harvested, washed, and stimulated with the gD peptides for 24 h in IFN ?
ELISPOT plates (Millipore) that have been previously coated with anti-
human IFN-? capture Abs in a humidified incubator at 37°C with 5% CO2.
The spot-forming cells were developed as described by the manufacturer
(BD Pharmingen) and counted under a stereoscopic microscope.
CD107 cytotoxicity assay
To detect cytolytic CD8?T cells recognizing gD peptides in freshly acti-
vated and in vitro activated PBMCs, we used the CD107a/b cytotoxicity
assay. Betts and colleagues (40, 42) recently described the CD107 assay as
an alternative cytotoxicity assay that is able to address some of the short-
comings of the51Cr release assay. The CD107 assay was performed as
described by Betts and colleagues (40, 42) with a few modifications. On the
day of the assay, unstimulated or in vitro gD peptide-stimulated PBMCs
(for 7, 14, or 21days) were incubated at 37°C for 5 to 6 h with BD Golgi
Stop (BD Biosciences), costimulatory anti-CD28 and anti-CD49d Abs (1
?g/ml), and 10 ?l of CD107a-FITC and CD107b-FITC. At the end of the
incubation period the cells were harvested into separate tubes and washed
once with FACS buffer then stained with PE-conjugated anti-human CD8
for 30 min. The cells were then washed again and analyzed using a
FACScan (BD Biosciences).
To evaluate whether a CD8?T cell response to peptide-pulsed or recom-
binant vaccinia virus (rVV)-HSV-infected target cells was statistically sig-
nificant, we used the Student t test to compare two sets of variables: 1)
mean IFN-? spots generated by CD8?T cells (from HSV-seropositive
humans or infected HLA-A*0201 mice) in response to JA2.1 cells pulsed
with peptide (relevant vs irrelevant) or infected with an rVV or HSV con-
struct (relevant vs irrelevant); or 2) mean IFN-? spots generated in re-
sponse to peptide-pulsed or rVV- or HSV-infected JA2.1 cells by CD8?T
cells from infected vs noninfected humans or mice. We used 2-by-2 con-
tingency tables to determine whether an association existed between pep-
tides with binding affinities of ?100 nM or ?100 nM and peptides that
were immunogenic in HSV seropositive human or infected HLA-A*0201
mice. The p values from the two-tailed Fisher exact test are reported.
In silico prediction and in vitro binding of potential HSV-1 gD
CD8?T cell epitope peptides to purified HLA-A*0201
The HLA-A*0201 allele, the prototypic member of the HLA-A2
supertype family, is predominant in the human population (26–
29). In addition, mice transgenic for this molecule are available
(26–28). We therefore decided to search the deduced HSV-1
(strain 17) gD amino acid sequence (including the signal sequence
indicated below by negative numbers) for potential HLA-A*0201
binding regions using BIMAS, SYFPEITHI, MAPPP, and MHC
Pred predictive computational algorithms. Based on these analy-
ses, 10 potential peptide epitopes with high predicted affinity to
HLA-A*0201 molecules were selected (Table I). Nine of the 10
Table I. Potential HLA-A*0201 restricted CD8?T cell epitopes predicted by BIMAS, SYFPEITHI, MAPPP, and MHCPred algorithms within the
HSV-1 (strain 17) gD sequencea
Peptide Sequence MWAA BIMASSYFPEITHI MAPPPMHCPredHLA-A*0201 Affinityb
gD?18 to ?10
gD?13 to ?5
gD?11 to ?2
aThe table shows the position of each peptide relative to the full length protein including the signal sequence, the amino acid (AA) sequence of each synthetic peptide (as
single letter code), their molecular weight (MW), the total number of amino acids, and the predicted IC50as calculated by BISMAS (www.bismas.cit.nih.gov/molbio.hla_bind),
SYFPEITHI (www.syfpeithi.de), MAPPP (www.mpiib-berlin.mpg.de/MAPPP), and MHCPred (http://www.mhc-pathway.net).
bIn vitro binding affinity to purified HLA-A*0201 molecules expressed as means of binding capacities (IC50nM) determined from two independent experiments.
428HUMAN CD8?T CELL EPITOPES ON HSV gD
by guest on June 18, 2013
gD peptide epitopes shared the HLA-A*0201-binding motifs:
leucine, isoleucine, or methionine at the second position and a
valine, alanine, leucine, methionine, or threonine at the ninth po-
sition. Based on the above computational algorithms, these gD
peptides, bearing putative antigenic and immunogenic HLA-
A*0201-binding epitope(s) are probably less constrained than
other parts of the gD molecule and are therefore more readily
accessible to proteolysis, an event that precedes T cell epitope
presentation in association with HLA molecules (26–29, 43).
Three of the 10 predicted epitopes are located within the gD
signal sequence (gD?13 to ?5, gD?11 to ?2, and gD?18 to ?10). Four
belong to the external N-terminal portion of gD (gD53–61, gD70–78,
gD95–103, and gD153–161). Three are adjacent to the hydrophobic
membrane anchor domain (gD224–232, gD253–262, and gD278–286).
Interestingly, none of the predicted peptides are within the hydro-
philic C-terminal cytoplasmic portion of gD, and all 10 peptides
are in nonglycosylated regions.
The 10 highest probability gD epitope peptides were synthe-
sized and screened for binding affinity to purified HLA-A*0201
molecules (Table I). The gD?18 to ?10, gD53–61, gD70–78, and
gD278–286peptides had the highest affinity binding to HLA-
A*0201 molecules (Kdaffinity values of 19–62 nM). Of the re-
maining six peptides, gD153–161, gD224–232, and gD253–26showed
medium affinity to HLA-A*0201 molecules (Kdaffinity values of
2002–4891 nM), whereas gD?13 to ?5, gD?11 to ?2, and gD95–103
had low affinity (Kd? 50,000 nM). Overall, these results suggest
that seven of the 10 gD peptides bind to HLA-A*0201, with gD53–61,
gD70–78, and gD278–286being the highest binders.
High-affinity gD peptides stabilized HLA-A*0201 molecules on
We next performed a stabilization assay of HLA-A*0201 mole-
cules on class I HLA-transfected B ? T hybrid cell lines (T2lines).
This assay measures the increase of HLA-A*0201 molecules on
the surface of T2cells, which is normally at a low level (44, 45).
Each gD peptide was tested at a concentration of 60 ?M (not
shown) and then titrated in serial 2-fold dilution steps (Fig. 1). Of
the four peptides previously showing the highest affinity binding to
purified HLA-A*0201 molecules, gD53–61, gD70–78, and gD278–286
peptides significantly increased levels of HLA-A*0201 molecules
on the surface of the T2cells (p ? 0.005) in a dose-dependent
manner (Fig. 1). In contrast, the remaining seven peptides pro-
duced no significant stabilization of HLA-A*0201 molecules de-
tectable by FACS on the surface of T2cells (Fig. 1).
gD epitope-specific, IFN-?-producing CD8?T cells detected in
healthy HLA-A*0201-positive, HSV-seropositive individuals
We next assessed the ability of high and low affinity peptides to
recall HSV-specific CD8?T cells in a total of 61 HLA-A*0201-
positive and HLA-A*0201-negative healthy individuals (Table II).
All individuals had well-defined clinical histories with one or no
episodes of recurrent disease per year (i.e., “protected asymptom-
atic” or “low-recurrent” disease), were healthy, hepatitis seroneg-
ative, and HIV seronegative. At the time of the study, none of them
had experienced any clinical ocular or genital herpes symptoms for
at least 1 year. In addition, the PBMCs from all 61 individuals
produced a positive T cell response to the mitogens LeuA
We used fresh peripheral blood-derived CD8?T cells isolated
directly ex vivo to minimize artifacts that could arise from in vitro
restimulation of CD8?T cells. The number of gD epitope-specific
IFN-?-producing CD8?T cells was determined by ELISPOT as-
say. Analysis revealed that 95 or more spots per 105CD8?T cells
by gD peptides on the surface of T2cells: A, T2cells
(3 ? 105) were incubated with serial dilutions of the
indicated gD peptide as described in Materials and
Methods. Then cells were stained with FITC-conjugated
anti-HLA-A2 mAb (BB7.2). The graph represents the
percentage of MFI increase of HLA-A*0201 molecule
expression on the surface of T2cells following incuba-
tion with different concentrations of gD peptides. The
percentage of MFI increase was calculated as follows:
percentage of MFI increase ? (MFI with the given pep-
tide ? MFI without peptide)/(MFI without peptide) ?
100. Error bars show SD for three independent
Stabilization of HLA-A*0201 molecules
Table II. Cohort of individuals used in this study
Subject Level Characteristic Results for All Subjects (n ? 61)
Age (Median/range)31 (18–63 years)
HSV status (Number/percentage)
HSV-1- and HSV-2 positive
429The Journal of Immunology
by guest on June 18, 2013
gave a 99.5% probability for defining a positive response, and 95
spot-forming units (SFU) per 105CD8?T cells was therefore used
as the cutoff point for subsequent analyses. Of the 560 separate
ELISPOT wells used to screen the 27 HSV-seronegative individ-
uals, only 11 wells (?2%) exceeded 95 SFU per 105CD8?T cells.
Of the 1386 separate ELISPOT assays used to screen the seropos-
itive individuals, 478 (17%) had readings of ?95 SFU per 105
CD8?T cells. Overall, 32 of 33 HSV-seropositive individuals re-
sponded to at least one of the gD peptides (i.e., had 95 or more
spots per 105CD8?T cells).
Next, we compared the IFN-?-producing CD8?T cells from
HLA-A*0201-positive vs HLA-A*0201-negative seropositive in-
dividuals (HSV-1 and/or HSV-2) (Fig. 2A). Positive IFN-?-pro-
ducing CD8?T cell responses were assessed by comparison with
control wells without Ag. For all but one peptide (gD224–232), sig-
nificantly more CD8?T cells producing IFN-? were seen in HLA-
A*0201-positive compared with HLA-A*0201-negative individu-
als. The highest IFN-? response appeared to be recalled by the
gD53–61epitope peptide (p ? 0.0001; Fig. 2, A and B).
For each of the high-affinity peptides (gD53–61, gD70–78, and
gD278–286), significant IFN-?-producing CD8?T cell responses
were detected in 45–95% of HLA-A*0201-positive, HSV-seropos-
itive individuals. The highest frequency of IFN-?-producing
CD8?T cell responses was detected against gD53–61peptide
epitope (95%). In comparison, only 14–24% of individuals
showed positive T cell responses to the remaining low affinity pep-
tides (gD?13 to ?5, gD?11 to ?2, gD?18 to ?10, gD70–78, gD95–103,
gD153–161, and gD224–232). There was no difference in T cell re-
sponses detected in individuals seropositive for HSV-1, HSV-2,
and HSV-1/HSV-2 (data not shown). We then stimulated CD8?T
cells with high-affinity peptides (gD53–61, gD70–78, and gD278–286)
for 6 days and analyzed the T cell response. Fig. 2B shows IFN-?
producing CD8?T cells specific to three immunodominant pep-
tides detected in HLA-A*0201-positive but not in HLA-A*0201-
gD epitope-primed CD8?T cells displayed lytic activity
CD107a and CD107b are lysosomal-associated membrane glyco-
proteins that surround the core of the lytic granules in cytotoxic T
cells (40, 46). Upon TCR engagement and stimulation by Ags in
association with MHC molecules, CD107a/b are exposed on the
cell membrane of cytotoxic T cells. Thus, the measurement of
CD107a/b expression on the surface of CTL is used as a direct
assay for the epitope-specific CTL response (40, 42). To assess
individuals. A, Fresh peripheral blood-derived CD8?T cells isolated directly ex vivo were stimulated with individual gD peptides. The numbers of gD
epitope-specific IFN-?-producing T cells were determined by ELISPOT assay. B, IFN-? produced by CD8?T cells assessed in HLA-A*0201-positive and
HLA-A*0201-negative individuals. PBMCs were stimulated for 6 days in vitro with gD53–61, gD70–78, and gD278–286peptides and then stimulated for 24 h
with the respective gD peptide on ELISPOT plates; tests were performed in duplicate for each experiment. Each horizontal line represents the mean for
all the individuals (n ? 34 for HLA-A*0201-positive and n ? 27 for HLA-A*0201-negative individuals). The spots were developed as described in
Materials and Methods and calculated as follows: spot forming cells (SFC) ? [(mean number of spots in the presence of antigen) ? (mean number of spots
in the absence of stimulation)]. ?, p ? 0.05 between the HLA-A*0201-positive and HLA-A*0201-negative groups.
gD epitope-specific IFN-?-producing CD8? T cells detected in healthy HLA-A*0201-positive HSV-seropositive HLA-A*0201-positive
430 HUMAN CD8?T CELL EPITOPES ON HSV gD
by guest on June 18, 2013
whether gD epitope-specific CD8?T cells display lytic activity,
fresh PBMC-derived CD8?T cells from HLA-A*0201-positive
and HLA-A*0201-negative individuals were stimulated in vitro
with individual gD peptides and CD107a/b expression was exam-
ined by flow cytometry on gated CD8?T cells (Fig. 3A). gD53–61,
gD70–78, and gD278–286peptides induced ex vivo significant
CD107 a/b expression on CD8?T cells from healthy HLA-A*0201-
positive, HSV-seropositive individuals. Although, gD?18 to ?10,
gD?13 to ?5, gD?11 to ?2, gD224–232, and gD253–262also showed a
positive cytotoxicity as detected by CD107 degranulation cyto-
toxic assays, these responses might not be specific and there is a
possibility that these gD peptides, unable to stabilize HLA-A*0201
on T2cells, might share specificities with human HLA-A2 sub-
types other than HLA-A*0201. Fig. 3B shows the in vitro FACS
results when cells from HLA-A*0201-positive individuals were
first stimulated with a heat-inactivated virus and then functional
cytolytic activity (CD107a/b/CD8?) was assessed against individ-
ual gD peptides. As expected, even after an in vitro restimulation
with an inactivated virus no CD8?T cells specific to the gD53–61,
gD70–78, and gD278–286peptides were detected in HLA-A*0201-
negative, HSV-seropositive individuals (Fig. 3C). There was no
CTL response against any peptides in individuals that were sero-
negative for HSV regardless of whether they were HLA-A*0201
positive or HLA-A*0201 negative (data not shown).
gD53–61, gD70–78, and gD278–286epitopes were naturally
processed and presented from native HSV gD in human
HLA-A*0201-positive target cells
Because the CD8?CTLs were generated by in vitro stimulation with
peptides to ascertain whether they recognized the processed products
of endogenously synthesized HSV-derived protein and not just pep-
tides, we analyzed the cytolytic activity of CD8?T cell lines gener-
ated by each gD peptide against HLA-A*0201-positive target cells
infected with HSV-1, HSV-2, VVgD, or an empty vaccinia virus
(control; not expressing gD). Fig. 4 shows that CD8?T cell lines
specific to the gD53–61and gD278–286peptides undergo significant
CD107a/b degranulation when incubated with HLA-A*0201-positive
target cells infected with VVgD, HSV-1 (strain McKrae), or HSV-2
(strain 333) (p ? 0.005). In contrast, the gD70–78-specific T cell line
was able to undergo significant CD107a/b degranulation only when
incubated with HLA-A*0201-positive target cells infected with
HSV-1 or HSV-2 (p ? 0.005) but not with VVgD. This suggests that
HSV-infected target cells. As a control, no cytolytic activity was de-
tected when gD53–61-, gD70–78-, or gD278–286-specific CD8?T cell
lines were incubated with HLA-A*0201-positive target cells infected
with empty vaccinia virus (designated VVC (control) in Fig. 4).
Therefore, these gD53–61, gD70–78, and gD278–286-specific CTLs
played lytic activity. PBMCs from six different HLA-
A*0201-positive and HLA-A*0201-negative individu-
als were stimulated with the 10 gD peptides in the
presence of anti-CD28/49d, FITC-conjugated anti-
CD107a and b, and Golgi Stop for 6 h. All peptides
were used at a final concentration of 20?M. A, The
graph represents the mean ? SD of the percentage of
CD107a/b and CD8?T cells in the presence of the gD
peptide subtracted from the percentage of cells without
peptide. B, Representative FACS analysis of CD107
expression after PBMC stimulation with respective gD
peptides, anti-CD3, or untreated (None). Events shown
are gated on the CD8?population and represented as
dot plotd for CD8 and CD107. C, CD8?cytotoxic T cell
activity assessed against gD53–61, gD70–78, and gD278–286
peptides in HLA-A*0201-positive and HLA-A*0201-
negative individuals. ]. ?, p ? 0.05 between the HLA-
A*0201-positive and HLA-A*0201-negative groups.
gD epitope-primed CD8?T cells dis-
431The Journal of Immunology
by guest on June 18, 2013
were specific for naturally processed products of endogenously syn-
thesized gD protein, not just exogenous peptides.
High frequency of gD53–61, gD70–78, and gD278–286
epitope-specific CD8?T cells detected in HLA-A*0201-positive,
To obtain an objective enumeration of gD epitope-specific CD8?
T cells, we constructed HLA-A*0201 tetramers incorporating all
but one gD peptide (i.e., gD?13 to ?5). For technical reasons we
were unable to produce a tetramer specific to the gD?13 to ?5pep-
tide epitope. Using peptide/PE-labeled HLA-A*0201 tetramers to-
gether with an FITC-conjugated mAb specific to human CD8?T
cells, we directly visualized the number of circulating CD8?T
cells specific to each peptide ex vivo (i.e., without in vitro stimu-
lation) and after in vitro stimulation. For each of the high-affinity
peptides gD53–61, gD70–78, and gD278–286, a significantly higher
percentage of tetramer?CD8?T cells was detected after in vitro
stimulation of the PBMCs of HLA-A*0201-positive, HSV-sero-
positive individuals (Fig. 5) compared with HLA-A*0201-nega-
tive, HSV-seropositive individuals. Similar to the IFN-?-produc-
ing CD8?T cell and CD8?/CD107a/b responses described in
Figs. 2A and 3, the highest frequency of tetramer?CD8?T cells
was detected against the gD53–61peptide epitope (6.2%). Despite
repeated attempts, the remaining six gD peptide/HLA-A*0201 tet-
ramers consistently detected a low percentage of CD8?T cells. To
enumerate the frequency of gD peptide-specific CD8?T cells in
unstimulated PBMCs, freshly ex vivo sorted CD8?T cells were
analyzed for the percentage of tetramer?CD8?T cells. A high
frequency of gD53–61- (7.2%) but not gD70–78- and gD278–286-
specific CD8?T cells was detected ex vivo in HLA-A*0201-pos-
itive HSV-seropositive individuals. Collectively, these results
show a high frequency of gD53–61-, gD70–78-, and gD278–286-spe-
cific CD8?T cells from healthy HLA-A*0201-positive HSV-se-
ropositive individuals, suggesting that these peptides contain func-
tional HLA-A*0201-restricted CD8?T cell epitopes generated
during HSV infection.
nodominant CD8?T cell epitope peptides recognized
infected target cells expressing native gD. gD53–61,
gD70–78, and gD278–286peptide-specific CD8?T cell
lines were generated from HLA-A*0201-positive indi-
viduals and their cytotoxic activity was tested (i.e. ca-
pacity to degranulate) in the presence or absence of tar-
get cells infected with HSV-1, HSV-2, or with VVgD.
Target cells infected with an empty vaccinia (VVC)
were used as negative controls. Results are representa-
tive of three independent experiments. ?, p ? 0.05.
CD8?T cell lines generated by immu-
epitope-specific CD8?T cells detected
in HLA-A*0201-positive HSV-sero-
positive individuals. In vitro activated
PBMC and freshly purified CD8?T
cells were analyzed for the frequency
of CD8?T cells recognizing gD
peptide/tetramer complexes. Tetramer
analysis of PBMCs from HLA-A*
positive individuals that have been
stimulated in vitro with heat inac-
tivated HSV-1 (PFU ? 5). Results
shown are representative of seven
432HUMAN CD8?T CELL EPITOPES ON HSV gD
by guest on June 18, 2013
Induction of gD peptide-specific CD8?T cells in HSV-1- and
HSV-2 infected HLA-A*0201 Tg mice
To ascertain the lack of cross-reactivity of gD-specific CD8?T
cell responses with self-derived or other pathogen-derived epitopes
as has been reported for some heterologous infections (47–49), we
assessed the percentage of tetramer?CD8?T cells (Fig. 6A) as
well as the frequency of IFN-? producing CD8?T cells (Fig. 6B)
specific to each gD peptide in HLA-A*0201 Tg mice infected with
HSV-1 (strain McKrae). HLA-A*0201 Tg mice infected with
HSV-1 developed high frequencies of CD8?T cells specific to
gD18 to ?10, gD53–61, gD70–78, gD95–103, gD153–161, gD224–232, and
gD278–286detected by either tetramer (Fig. 6A) or IFN-?–ELISPOT
(Fig. 6B). Unlike HLA-A*0201-positive, HSV seropositive hu-
mans, HLA-A*0201 Tg mice infected with HSV-1 did mount
CD8?T cell responses against cryptic gD?18 to ?10, gD95–103,
gD153–161, and gD224–232epitopes, which could reflect a higher
level of infection in HLA-A*0201 Tg mice compared with HLA-
A*0201-positive humans. However, in the HLA-A*0201 Tg mice
Table III. Comparative analysis of the sequences of HSV-1 immunodominant CD8?T cell gD epitopes
within and between HSV-1 and HSV-2 strains
Virus and Strain (GenBank Accession No.) gD53–61a,b
Strain 17 (Q69091)
Strain Angelotti (P36318)
Strain Patton (P57083)
Strain HZT (P06476)
Molybdopterin biosynthesis protein (Q51CU7)
gD precursor (P03172)
Virion gD (Q69467)
aThe nonidentical amino acids between HSV-1 and HSV-2 are bolded and underlined.
bThe nonidentical amino acids between HSV-1 strains are bolded, underlined, and italicized.
epitopes in HLA-A*0201 Tg mice following infection
with either HSV-1 or HSV-2. HLA-A*0201 Tg mice
were infected with HSV-1 (strain McKrae) at 5 ? 105
PFU or with HSV-2 (strain 333) at 106PFU. Spleen-
derived CD8?T cells were isolated 14 days postinfec-
tion and the percentage of gD-specific CD8?T cells
was determined by tetramer staining (A) or they were
exposed to JA2.1 target cells that had been pulsed with
each of the gD peptides (10 ?M) in a CD107a/b de-
granulation cytotoxic assay (B and C). Error bars show
SD for seven independent experiments. ?, p ? 0.05.
Immunogenicity of CD8?T cell gD
433The Journal of Immunology
by guest on June 18, 2013
infected with HSV-1, the highest frequency of CD8?T cells ap-
peared to be directed against the gD53–61, gD70–78, gD153–161,
gD224–232, and gD278–286peptides. In addition, CD8?T cell re-
sponses specific to the immunodominant gD53–61, gD70–78, and
gD278–286peptides were detected in HLA-A*0201 Tg mice fol-
lowing infection with either HSV-1 (strain McKrae) or HSV-2
(strain 333) Fig. 6C). This is consistent with the sequences of these
three epitopes being highly conserved among and between HSV-1
and HSV-2 strains (Table III).
This study identified three human CD8?CTL epitopes from
HSV-1 gD, a glycoprotein that produces protective immunity in
both animal models and humans (3, 4, 7, 8, 10, 50). These new gD
epitopes displayed high-affinity binding to purified HLA-A*0201
molecules, stabilized HLA-A*0201 molecules on target cells, re-
called CD8?CTLs in HSV-1 and HSV-2 seropositive individuals
as well as in HLA-A*0201 Tg mice infected with either HSV-1 or
HSV-2, and were naturally processed from native HSV-1/2 gD in
HLA-A*0201-positive target cells.
In humans, HSV-specific CD4?and CD8?T cells play a crucial
role in controlling both primary and recurrent infections (51, 52).
Results from a number of studies have documented HSV gD as
being a target for both CD4?and CD8?CTLs. In the late 1980s
and early 1990s, Zarling and coworkers (53, 54) generated several
human CD4?CTL clones from HSV-1-seropositive individuals by
stimulating PBLs with a rVV expressing HSV-1 gD. Five of these
CD4?CTL clones lysed autologous HSV-1-infected LCLs and
their cytotoxicity was restricted to HLA class II molecules. Later,
it was demonstrated that some of these HSV-1-specific CTL clones
were directed against both gD-1 and gD-2 by using target cells
infected with wild-type HSV strains, a glycoprotein C deletion
mutant of HSV-1, and a recombinant HSV-1 ? HSV-2 virus (55,
56). In another study, purified HSV-1 gD expressed in mammalian
cells stimulated the proliferation of and the IL 2-production by the
PBLs of HSV-seropositive individuals, indicating the presence of
HSV gD-specific memory T cells (57). In addition, T cell clones
generated by the stimulation of PBL with HSV-1 were found to
proliferate in response to gD-1 in the absence of exogenous IL 2
and to lyse HSV-1 or both HSV-1/-2-infected autologous target
cells (53). HSV-specific cytotoxic CD4?and CD8?T cell clones
have been recovered ex vivo from the HSV-2 lesions of five pa-
tients by Koelle and coworkers (58, 59). By isolating HSV-specific
CD8?CTL clones from a patient with recurrent genital herpes,
Burke and coworkers (60) also reported one CD8?T cell clone
that lysed vaccinia virus/gD2-infected target cells. Cunningham
and coworkers (61, 62) have also reported that HSV gD is a major
target for both CD4?and CD8?CTLs using human epidermal
keratinocytes as targets. Although, the above findings demon-
strated gD as a major target for human HSV-specific CD4?and
CD8?CTLs, no specific gD minimal epitopes were defined.
Therefore, to our knowledge the present study reports the first
three immunodominant HLA-A*0201-restricted human CD8?T
cell gD epitopes. This does not imply, however, that these are the
only human CD8?T cell gD epitopes.
High-performing predictive computational algorithms have
been available for more than a decade; however, their value in
terms of actual epitope finding has only now been extensively
studied (11, 24–28, 63, 64). Many immunological screens have
been recently advanced to validate predicted human CD8?T cell
epitopes, including overlapping synthetic peptides, binding affinity
to purified HLA molecules, tetramer assays, cell membrane HLA
stabilization assays, and CD8?T cell cytotoxic assays (65–68).
When used individually, each screen is not sufficient for identify-
ing functional CD8?T cell epitopes (32, 65, 67, 68). However,
different combinations of these screens are usually successful (69–
71). In this study we supported our predictive computational al-
gorithms by multiple immunological parameters, including in vitro
assays of binding affinity to purified HLA-A*0201 molecules, cell
surface stabilization of HLA-A*0201 molecules on T2cells, func-
tional ex vivo and in vitro CD8?T cells assays, and tetramer
staining of individual CD8?T cells. Using these multiple screens,
we identified three high-affinity gD peptides, gD53–61, gD70–78,
and gD278–286, that were able to induce functional IFN-? produc-
ing-CD8?T cells, display cytotoxic activity, and recognize native
epitopes on HSV-infected target cells. Moreover, the frequency of
CD8?T cells in HLA-A*0201-positive CD8?T cells was higher
for the gD53–61, gD70–78, and gD278–286peptides compared with
HLA-A*0201-negative CD8?T cells, suggesting HLA-A*0201
restriction of these responses. Although gD?18 to ?10, gD?13 to ?5,
gD?11 to ?2, gD224–232, and gD253–262also showed a positive cy-
totoxicity detected by CD107 degranulation cytotoxic assays, these
responses might not be specific nor HLA-A*0201 restricted. In-
deed, CD107a/b assays were performed using PBMCs and there is
a possibility that these gD peptides, unable to stabilize HLA-
A*0201 on T2 cells, might share specificities with human HLA-A2
subtypes other than HLA-A*0201. Because of the relative rarity of
circulating HSV-specific memory T cells compared with those for
CMV and EBV (72), it is surprising that tetramer?CD8?T cells
specific to the gD53–61epitope were consistently detected ex vivo
in HSV-seropositive, asymptomatic individuals (?1 year after
their last recurrence). This may indicate that HSV-infected patients
have a high frequency of circulating gD53–61-specific T cells and
that this epitope might be the immunodominant HLA-A2.1-re-
stricted epitope of gD, as is also attested by the high level of IFN-?
ELISPOT and CD107a/b degranulation cytotoxic functional as-
says. Although our approach to epitope identification in this study
was tailored specifically for HLA-A*0201-restricted epitopes, it
represents a model system that could be applied to any HLA class
I haplotype and to any other herpes structural or regulatory protein.
The minimum requirements needed to use this approach are: 1)
existing sequence data for the proteins in question; 2) access to the
genome (DNA or RNA) of the pathogen for the purpose of gen-
erating rVV vectors; 3) HLA-A*0201-positive HSV, HSV-sero-
positive individuals; and 4) HLA Tg mice. Therefore, our ap-
proach to epitope identification as well as to the testing of epitopes
for their human T cell antigenicity should be nearly universal re-
gardless of the protein studied.
Even though the findings in this report can be enlightening, hu-
mans are not immunologically naive and they often have memory
T cell populations that can cross-react with and respond to other
infectious agents. These cross-reactive T cells can become acti-
vated and modulate the immune response and outcome of subse-
quent heterologous infections, a phenomenon termed heterologous
immunity (49). Therefore, we cannot exclude the possibility that
some HSV-specific CD8?T cells identified in this study are cross-
reactive with self-or other pathogen-derived Ags. Such scenarios
have been reported in murine heterologous infection (47, 48) and
may be true in humans, as shown for CD4?T cell responses to
CMV (47–49). To investigate the possibility of heterologous T
cell responses against the gD53–61, gD70–78, and gD278–286peptide
epitopes identified in this study, the epitope mapping was extended
to pathogen-free HLA-A*0201 Tg mice that were infected with
either HSV-1 or HSV-2. The results showed that in these HLA-
A*0201 Tg mice, CD8?T cell responses mapped to gD53–61,
gD70–78, and gD278–286epitopes as recognized by human CD8?T
cells. Although, the profile of responding peptides appears differ-
ent between HLA-A*0201-positive humans and HLA-A*0201 Tg
434HUMAN CD8?T CELL EPITOPES ON HSV gD
by guest on June 18, 2013
mice (Fig. 6), all three immunodominant peptides (gD53–61, gD70–78,
and gD278–286) detected in HLA-A*0201-positive humans were
also positive in HLA-A*0201 Tg mice. The Tg mice also re-
sponded to additional epitopes including gD?18 to ?10, gD95–103,
gD153–161, and gD224–232. This nonsurprising discrepancy might
be due to the following: 1) the class I molecule in HLA-A*0201
Tg mice is composed of human ?1 and ?2 domains and the mouse
?3 domain to allow mouse CD8 binding, which could affect the
presentation of some epitope peptides; 2) the amount of HSV used
to infect the mice may greatly exceed the amount for a typical
primary human infection, which might differentially affect CD8?
T cell stimulation for some epitopes; and 3) the processing/pre-
sentation machinery of the HSV virus in mice and humans is
The ability of CD8?T cells generated by gD53–61, gD70–78, and
gD278–286epitopes to kill target cells endogenously expressing the
gD protein (i.e., infected with HSV or VVgD) is a critical param-
eter, especially given the observation that CTLs induced by pep-
tide in vitro may not kill targets expressing the endogenous pro-
tein. However, a difference in the level of gD expression by LCL
target cells might occur following VVgD, HSV-1, and HSV-2 in-
fection. This could affect the functional presentation of some gD
epitopes by infected target LCLs as detected by CD107a/b degran-
ulation and IFN-? ELISPOT assays. Indeed, although the gD70–78-
specific CD8?T cell line efficiently recognized HSV-1- and HSV-
2-infected target cells, its level of cytotoxicity was slightly lower
compared with gD53–61- and gD278–286-specific T cell lines. In
contrast, the same gD70–78-specific CD8?T cell line failed to
recognize the same LCL target cells when infected with VVgD.
Thus, although the gD70–78epitope appeared to be efficiently pre-
sented by HSV-1- and HSV-2-infected LCL target cells, this did
not appear to be the case in VVgD-infected LCL target cells. One
possibility to account for this difference might be that the gD ex-
pression level in VVgD-infected LCLs, although sufficient for ef-
ficient presentation of other gD epitopes, was for as yet undeter-
mined reasons insufficient for efficient presentation of the gD70–78
epitope. It is also possible that the correct presentation of this
epitope requires one or several factors produced in HSV-infected
cells, because this epitope was efficiently presented in HSV-1- and
HSV-2-infected LCLs. Although some gD-expressing cell lines
are available, HLA-A2.1-positive target cell lines constitutively
expressing gD are not currently available. In the future, we hope to
construct such a cell line. Thus, it is very likely that the gD53–61,
gD70–78, and gD278–286epitopes detected in humans were not
cross-reactive but were induced following HSV-1/HSV-2 infec-
tion. This does not imply excluding the involvement of other in-
fectious agents in shaping HSV T cell epitope-specific responses.
The vast majority of the world’s human population is infected
with HSV-1 and/or HSV-2 and might potentially benefit from ther-
apeutic vaccination approaches designed to boost HSV-specific
cellular immunity. Although the primary goal of this study was to
identify human CD8?T cell epitope peptides on HSV gD, a sec-
ondary but important goal is to develop the knowledge of herpes T
cell epitopes required for the development of a totally synthetic,
self-adjuvant lipopeptide human vaccine. We now plan first to con-
struct lipopeptide vaccine candidates incorporating the CD8?CTL
epitopes identified in the present study together with the human
CD4?T helper epitopes that we (Ref. 1 and X. Zhang, F. A.
Castelli, M. Y. Wu, I. Bettahi, D. Carpenter, A. Mohebbi, A. B.
Nesburn, S. L. Wechsler, B. Maille ´re, and L. BenMohamed, sub-
mitted for publication) and others (A. L. Cunningham, unpublished
observations) have recently identified. Such a combination would
produce CD4?CD8?chimeric lipopeptide candidate vaccines simi-
lar to those we have recently described in mice against ocular and
genital herpes infection (2). Secondly, we plan to study the safety,
immunogenicity, and protective efficacy in humans of a CD4?CD8?
chimeric epitope lipopeptide candidate vaccines.
Recently, we reported four immunodominant H2d-restricted
CD4?T cell epitopes on HSV-1 gD, among which is the peptide
gD49–82, a naturally processed epitope that protects H2dmice
against lethal ocular HSV-1 challenge (1). We have also recently
found that the gD49–82epitope represents an immunodominant
HLA-DR*0101 and HLA-DR*0104-restricted human CD4?T
cell epitope (X. Zhang et al. submitted for publication). Interestin-
gly, the gD53–61sequence, one of the three immunodominant hu-
man CD8?T cell epitopes identified in this study, is completely
contained within the gD49–82sequence. Therefore, gD49–82har-
bors at least two human epitopes, the gD53–61CD8?T cell epitope
and a CD4?T cell epitope and, as such, this gD region represents
a good candidate for inclusion in a CD4?and CD8?T cell
epitope-based human vaccine.
A question of practical importance is the translation of the cur-
rent immunological findings into the development of an epitope-
based vaccine for a genetically heterogeneous human population.
Although the high degree of HLA polymorphism is often pointed
to as a major hindrance to the use of epitope-based vaccines, this
constraint can be dealt with through the inclusion of multiple su-
pertype-restricted epitopes recognized in the context of diverse
related HLA alleles and by designing mixtures of peptide-based
vaccines with higher epitope densities (2, 3, 7, 8, 11, 24, 25).
Broad population coverage can be established providing that
epitopes corresponding to multiple HLA supertype families are
incorporated into the vaccine. Sette and Sidney (73) defined
nine HLA class I supertypes that provide an almost perfect cov-
erage (?99%) of the entire repertoire of HLA class I molecules.
Because the HLA-A2 supertype family is present in ?50% of the
general population (regardless of ethnicity), the CD8?T cell
epitopes identified in this study should prove to be useful in vac-
cine trials. To maximize efficacy, a multiepitope-based herpes vac-
cine should probably include several T cell epitopes from several
different structural glycoproteins and regulatory proteins, each
chosen to represent at least the HLA supertypes known to provide
recognition in a large proportion of the global population regard-
less of race and ethnicity. Hence, our laboratory is trying to iden-
tify such HLA-promiscuous T cell epitopes.
CD8?T cell infiltrates appear to correlate with HSV clearance
from mucocutaneous lesions (52). However, pathogens such as
HSV that establish a latent infection have evolved a variety of
immune evasion tactics to avoid being detected by the host’s im-
mune system. Among the central issues in the immunobiology of
HSV is its ability to evade the cellular immune system control and
periodically reactivate from latently infected sensory neurons (74–
78). Unlike HIV and other pathogens, antigenic variation is not
used by HSV as a mechanism to escape cell-mediated immune
surveillance. Rather, HSV has developed alternative mechanisms
in which specific viral genes inhibit antigenic processing and pre-
sentation to T cells, including CD8?T cells (78–80). The HSV
immediate-early protein ICP47 blocks the peptide transporters
(TAP1/TAP2) associated with Ag processing (74, 76, 81). This
decreases the access of antigenic peptides into the MHC pathway,
preventing the transport of HLA molecules to the surface of APCs
(74, 80, 82). The product of the US1 gene (76, 80) and the virion
host shutoff (vhs) or UL41 gene rapidly degrade host cell mRNA,
contributing to decreased synthesis of new HLA class I molecules
(83). All of these mechanisms might render HSV-infected neurons,
fibroblasts, keratinocytes, LCL/dendritic cells, and other mucocu-
taneous target cells insensitive to lysis by infiltrating CD8?CTLs.
Therefore, these powerful immune evasion mechanisms must be
435 The Journal of Immunology
by guest on June 18, 2013
taken into account in future vaccine strategies against herpes. Their
effect on epitope processing/presentation might be counteracted by
IFN-? or other treatments (e.g., acyclovir), thus providing a better
role for CD8?T cells in the cellular immunity to HSV (83, 84).
In conclusion, in this article we report three immunodominant
HLA-A*0201-restricted epitopes derived from the HSV-1 gD that
recall CD8?CTL responses in HLA-A*0201-positive, HSV-sero-
positive individuals and in HSV infected HLA-A*0201 Tg mice.
These functional CD8?T cell epitopes shared high sequence ho-
mology between and within HSV-1 and HSV-2 strains. Hence,
they should be considered in the design of effective immunother-
apeutic and immunoprophylactic strategies against HSV-1 and
HSV-2 in humans.
We thank Amy K. Stout from the National Institutes of Health Tetramer
Facility for providing the Tetramers, Dr. Francois Lemonnier (Pasteur In-
stitute) and Dr. Don J. Diamond (City of Hope) for providing the HLA-
A*0201 transgenic mice, and Dr. David Koelle for providing the vaccinia
viruses used in this study.
The authors have no financial conflict of interest.
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