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Journal of Virology, September 1998, p. 7476-7483, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by
CD4 T Cells Infiltrating Human Genital Herpes Lesions
David M.
Koelle,1,2,3,*
Jeannine M.
Frank,2,3
Matthew L.
Johnson,2 and
William W.
Kwok4
Department of
Medicine1 and
Department of Laboratory
Medicine,2 University of Washington,
Seattle, Washington 98105;
Fred Hutchinson Cancer Research
Center, Seattle, Washington 981043; and
Virginia Mason Research Center, Seattle, Washington
981014
Received 16 March 1998/Accepted 19 May 1998
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ABSTRACT |
The local cellular immune response to herpes simplex virus (HSV) is
important in the control of recurrent HSV infection. The antiviral
functions of infiltrating CD4-bearing T cells may include cytotoxicity,
inhibition of viral growth, lymphokine secretion, and support of
humoral and CD8 responses. The antigens recognized by many HSV-specific
CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells
recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA
are frequently recovered from herpetic lesions. Expression cloning with
this region of DNA now shows that tegument protein VP22 and the viral
dUTPase, encoded by genes UL49 and UL50,
respectively, are T-cell antigens. Separate epitopes in VP22 were
defined for T-cell clones from each of three patients. Reactivity with
the tegument protein encoded by UL21 was identified for an
additional patient. Three new epitopes were identified in VP16, a
tegument protein associated with VP22. Some tegument-specific CD4
T-cell clones exhibited cytotoxic activity against HSV-infected cells.
These results suggest that herpes simplex tegument proteins are
processed for antigen presentation in vivo and are possible candidate
compounds for herpes simplex vaccines.
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INTRODUCTION |
Cellular immune responses are
required to limit the severity of recurrent herpes simplex virus (HSV)
infections in humans (38). HSV-specific CD4 and CD8 T cells
infiltrate herpetic lesions (16, 17, 19). In vitro, some
HSV-specific CD4 T cells are cytotoxic toward virus-infected cells
(41, 42). HSV-specific CD4 T cells make large amounts of
gamma interferon (7), which may overcome HSV-mediated HLA
class I downregulation and permit lysis of HSV-infected cells by CD8
cytotoxic T lymphocytes (CTL) (35). Gamma interferon also
upregulates HLA class II on HSV-infected keratinocytes to allow
recognition by cytolytic CD4 T cells (26) and has direct
antiviral effects (13).
The antigenic specificity of HSV-specific CD4 T cells in infected
humans is incompletely defined. CD4 T-cell responses in peripheral
blood mononuclear cells (PBMC) to HSV envelope glycoproteins B, C, D,
and H have been detected by bulk culture, limiting dilution, or clonal
analyses (22, 26, 36, 44). PBMC-derived CD4 T-cell clones
(TCC) commonly recognize envelope glycoproteins (43, 44),
and bulk cultures of PBMC-derived HSV-specific CD4 CTL efficiently
recognize glycoprotein-expressing target cells (26).
However, we determined that only a minority of lesion-infiltrating CD4
TCC recognize glycoprotein B, C, or D (17), and bulk
cultures of lesion-infiltrating T cells show reactivity with
glycoprotein B or D for only 50 to 70% of donors (19). We
therefore undertook studies to define additional HSV T-cell antigens.
The region near 0.7 map units on the HSV type 2 (HSV-2) genome was
identified as rich in HSV-2 type-specific T-cell antigens (17). The unique long (UL) 48 gene, encoding
tegument protein VP16, contains at least three T-cell epitopes
(14) but does not account for all the antigenicity in this
region (17). Expression cloning with subgenomic DNA from
this region initially identified VP22 (encoded by UL49) and
dUTPase (encoded by UL50) as T-cell antigens. Expression
cloning with full-length viral DNA independently showed recognition of
VP22 by a lesion-derived TCC from a second donor, and local
VP22-specific responses were also detected for a third donor at bulk
and clonal levels. Three separate epitopes were identified in VP22.
Reactivity with an additional tegument protein, UL21, was
also detected. VP22 is tightly associated with VP16 in the virion
tegument as well as within infected cells (10). We therefore
extended our previous studies with VP16 and identified three additional
epitopes recognized by lesion-infiltrating T cells.
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MATERIALS AND METHODS |
Viruses and cells.
HSV-1 strain E115 (34), HSV-2
strain 333 (15), and intertypic recombinant viruses (IRV)
RS1G31 (29), DX32 (31), and RP-2 (17)
were grown and titered in Vero cells (20). Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL) included both
autologous cell lines created from donors with genital herpes as
previously described (20) and the characterized lines AMAI (homozygous for HLA DPB1*0402), HOM2 (homozygous for HLA DQB1*0501), MAT (homozygous for HLA DQB1*0201), and ARENT (homozygous for HLA
DPB1*2001) (4).
HSV-specific T cells were obtained after approval by the Institutional
Review Board. Most clones were derived without secondary in vitro
stimulation with antigen. Donors 1 through 4 are numbered as previously
described (17), and donors 1, 2, and 4 were the sources of
lesion-derived clones 1.L3D5.10.8, 2.3, and 4.2E1, respectively; clones
2.3 and 4.2E1 have been described previously (17).
Additional lesion-derived clones came from donor 5, from whose second,
third, and fourth lesion samples (each separated by 1 year) clones
ESL2.20, ESL3.334, ESL4.34, and ESL4.9 were derived. Other clones, such
as RH.13 and KM.7, were derived from donors 6 and 7. Clones 2.3, 4.2E1,
ESL2.20, RH.13, and KM.17 were derived directly from herpetic vesicle
fluid (16). To derive CD4 TCC ESL4.9, biopsy of a recurrent
genital HSV-2 lesion (day 3 of symptoms) was performed and bulk
lesion-infiltrating cells were expanded with phytohemagglutinin and
interleukin 2 (Schiaperelli Biosystems, Columbia, Md.) in the presence
of acyclovir as described elsewhere (19). After 16 days,
cells were cloned at 1 cell/well (16). Previously described
VP16-specific clones 1A.B.25, ESL3.334, and ESL4.34 (8, 14,
18) were similarly derived from bulk cultures.
Some clones were derived by using secondary in vitro stimulation with
antigen. To derive additional TCC from donor 1 (
17),
phytohemagglutinin-driven bulk cultures were prepared from each
of four
2-mm biopsy specimens (day 5 of symptoms) obtained 6 years
after the
occurrence of the lesion from which clone 1A.B.25 (mentioned
above) was
derived. After 16 days, 1.5 × 10
6 bulk lymphocytes
from one biopsy culture were stimulated with
10 µg of HSV-2 VP22,
amino acids 105 to 190 (VP22 105-190)/ml
(see below) and an equal
number of autologous irradiated (3,300
rads) PBMC in 2 ml of T-cell
medium (
16). Interleukin 2 (32
U/ml) was added starting on
day 6. TCC 1.L3D5.10.8 was isolated
from this line on day 12 as
described elsewhere (
16). To create
PBMC-derived TCC SB.17
and BM.17, 1.5 × 10
6 PBMC of HSV-2-seropositive
donors 8 and 9 were stimulated for
12 days with 4 µg of
baculovirus-derived full-length VP16/ml in
24-well plates; responding
cells were cloned at 1 cell/well. TCC
and lines were used 10 to 14 days
after the last stimulation.
All cell lines were negative for mycoplasmas except ARENT. ARENT was
initially positive for mycoplasmas by DNA probe test
(Genprobe, San
Diego, Calif.) and was treated with ciprofloxacin
at 10 µg/ml
(
12) for 2 weeks prior to utilization, with conversion
of
the test to negative.
Flow cytometry.
Flow cytometry used a combination of murine
monoclonal antibodies (MAb) to human CD4 (clone SFCI 12T4D11) and CD8
(clone SFCI 21Thy2D3, recognizing the 
chain of human CD8) (Coulter,
Hialeah, Fla.).
Immunoblotting.
Lysates of HSV-infected Vero cells were
prepared, electrophoresed through sodium dodecyl sulfate (SDS)-10%
polyacrylamide gels, and transferred to a nitrocellulose membrane as
described elsewhere (1). Blots were blocked with
phosphate-buffered saline (PBS)-0.05% Tween 20-1% nonfat dried
milk. Antigen was detected by sequential incubation with a 1:100
dilution of MAb P43, specific for the UL49 gene product,
VP22 (11), affinity-purified goat anti-mouse immunoglobulin
M-peroxidase conjugate (Sigma, St. Louis, Mo.), and the TMB substrate
system (Kirkegaard and Perry, Gaithersburg, Md.) with three washes (5 min each) in PBS-Tween between each step.
Viral DNA libraries and cloning.
For subgenomic DNA, the
HSV-2 strain HG-52 BamHI w fragment was subcloned from the
BglII i fragment and gel purified. Viral DNA was digested
with SmaI, BamHI ends were blunted with Klenow DNA polymerase, and DNA fragments were purified by phenol extraction and alcohol precipitation. For whole-virus DNA, confluent Vero cells
were infected with HSV-2 strain HG52. Total nucleic acids from three
150-cm2 cell cultures were prepared by proteinase K
digestion, chloroform-phenol extraction, and isopropanol precipitation.
The resultant material was treated with RNAse H and was reextracted and
precipitated. Aliquots (1 µg) of HG52 DNA were digested with
SmaI or AluI, and 80% of these digests were
combined and recovered as described above for creation of expression
libraries.
Expression cloning used pUEX vectors (
5). pUEX1, -2, and -3 DNAs were linearized with
SmaI, dephosphorylated with calf
intestinal phosphatase, and gel purified. Approximately 100 ng
of
vector and 500 ng of DNA fragments were ligated, and 10% of
ethanol-precipitated reaction mixtures were used to transform
Escherichia coli DH10 Electromax (GIBCO) by electroporation
(BTX,
San Diego, Calif.) in 1-mm cuvettes. After 1 h of recovery
in
1 ml of SOC medium, portions were frozen as glycerol stocks (100
µl each), titered on ampicillin plates at 30°C (250 µl), or used
directly (250 µl) for protein induction to create fusion protein
libraries. Several thousand ampicillin-resistant colonies were
isolated
per transformation. To amplify genomic libraries, glycerol
stocks were
grown overnight at 30°C in 2× YT-ampicillin medium
and refrozen.
Confirmatory subcloning of VP22 105-190, U
L50 118-312, and
U
L50 118-250 was performed by isolating the 262-bp
SmaI-
StuI fragment
of U
L49, the
583-bp
SmaI fragment of U
L50, and the 397-bp
SmaI-
StuI
fragment of U
L50,
respectively. Fragments were cloned into the
appropriate linearized,
gel-purified pUEX vector, and protein
was expressed in
E. coli DH5
. Constructs were confirmed by sequencing.
Antigens.
Whole-virus preparations containing
108 to 109 PFU/ml were UV inactivated for 30 min (16) and used at a 1:100 final dilution. Peptides of
VP22 were synthesized as described elsewhere (18) and used
as stocks at 2 mg/ml in dimethyl sulfoxide. Peptides of
UL48, 13 amino acids long and overlapping by 4 amino acids, and VP16 of HSV-2, amino acids 1 to 416, and full-length VP16, both
expressed in baculovirus, were a kind gift of Rae L. Burke and Michael
Tigges, Chiron Corporation, Emeryville, Calif.
Bacterium-derived protein antigen expression was induced for 2 h
at 42°C in cells growing logarithmically (optical density
at 600 nm,
0.4 to 0.6) in 2× YT-ampicillin broth at 30°C. Protein
was purified
as described elsewhere (
27), omitting gel purification.
Bacterial cultures of 50 ml (libraries) or 5- to 10-ml cultures
(pools
and clones) yielded fine particulate suspensions in 200
to 400 µl of
PBS (Ca and Mg free). Protein concentrations were
determined by
bicinchoninic acid assay (Pierce, Rockford, Ill.)
after proteins were
solubilized in 1% SDS at 60°C for 10 min.
In some experiments,
heat-induced bacteria were washed with PBS
and PBS-10 mM EDTA, heated
to 56°C for 10 min, and washed in PBS
prior to use as antigens.
After identification of an active library of viral DNA, antigen
identification used 30 to 60 clones for subgenomic viral DNA
fragments
or 2,000 to 3,000 clones for full-length viral DNA.
For the less
complex library, 1-ml cultures of each clone were
processed as pools of
six to eight clones. Individual clones within
the active pool, and
confirmatory subclones containing known viral
DNA fragments, were
processed as 5-ml cultures. A combinatorial
method (
27) was
used to screen libraries from whole-virus DNA.
Glycerol stocks of
amplified libraries of transformed bacteria
were titered on ampicillin
plates; 20 to 30 colonies/well were
cultured overnight at 30°C in a
96-well plate in a rotating shaker.
Cultures were diluted 1:100 into
1-ml cultures, and fusion protein
synthesis was induced as described
above. Portions (400 µl) of
cultures were pooled row- and columnwise
for protein purification
and evaluation in lymphoproliferation assays.
If more than one
row and column were positive, wells at the
intersections of positive
rows and one positive column were used to
prepare protein from
5- to 10-ml cultures to definitively identify a
positive well.
Cultures (
n = 96 colonies) of bacteria
were derived from ampicillin
plates seeded with diluted broth from
positive wells. These were
evaluated as pools (of 12 bacterial
colonies) and then individual
clones.
Lymphocyte functional assays.
Triplicate proliferation assay
wells contained 104 cloned T cells, 105
irradiated (3,300 rads) PBMC, or 2.5 × 104 irradiated
(8,000 rads) EBV-LCL as antigen-presenting cells (APC) and antigen in
200 µl of T-cell medium (18) in 96-well U-bottom plates.
When heat-killed bacteria were used as the antigen, the equivalent of
105 CFU/well (prior to inactivation) was added and
gentamicin (20 µg/ml) was included. After 72 h, 1 µCi of
[3H]thymidine/well was added for 18 h, cells were
harvested, and incorporation of thymidine was evaluated by liquid
scintillation counting. Standard deviations were less than 10% of the
mean values. Results are reported as mean counts per minute or as
change in counts per minute (
cpm), equal to mean counts per minute
for the experimental antigen minus mean counts per minute for the control antigen. The control antigen was mock-infected cell lysate for
whole-virus antigens and pUEX2-derived 
-galactosidase for recombinant protein preparations. To determine the reactivity of
bulk-cultured lesion-derived T cells, fusion proteins or control -galactosidase was used at 10 µg/ml. Glycoproteins B and D and VP16 of HSV-2 were used at 1 µg/ml, and assays were performed as
described previously (19). To determine HLA-restricting
loci, HLA-DR-specific MAb L243 (32), HLA-DP-specific MAb
B7.21 (37), or HLA-DQ-specific MAb SPV-L3 (33)
was used as described previously (17).
Cytolysis assays were performed in triplicate by using 4-h
51Cr release as described elsewhere (
20). Target
EBV-LCL were infected
for 18 h with HSV-2 at a multiplicity of
infection of 30 or pulsed
with 1.0 µM peptide for 90 min prior to
washing as described elsewhere
(
21). The effector-to-target
ratio was 20:1. Spontaneous release
was less than 28%.
DNA sequencing.
Viral inserts in plasmids in bacteria
yielding active proteins were completely sequenced (Taq DyeDeoxy FS
kit; Perkin-Elmer ABI, Foster City, Calif.) in both directions by
starting with primers CATGGCTGAATATCGACGGT (5' end of
insert) and CTAGAGCCGGATCGATCCGGTC (3' end of insert) and
then using internal primers designed as required.
HLA typing.
HLA-DR and -DQ typing was performed at class II
alleles by serologic methods or at the DNA level by reverse dot blot
hybridization (25). HLA-DP typing was performed by
sequencing (HLA-DP kit; Perkin-Elmer ABI).
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RESULTS |
Fine localization of T-cell epitopes.
To reduce the complexity
of libraries for expression cloning, we selected a TCC-recognizing
antigen(s) partially mapped by using HSV-1 × HSV-2 IRV. We have
previously determined that HSV DNA near 0.7 map units encodes T-cell
antigens in addition to VP16. Epitope mapping for TCC 4.2E1 and 2.3 (17) was improved with IRV DX32 (Fig.
1). This HSV-2-based virus contains a
block of HSV-1 DNA near 0.7 map units (31). The
UL48 gene product has the HSV-2 phenotype, as shown
by reactivity with the HSV-2 type-specific, VP16-specific
(17) TCC 1A.B.25. The UL49 (Fig. 2) and UL50 gene products
(39, 40) also have an HSV-2 phenotype. The HSV-2 DNA
present in IRV DX32 therefore includes UL48,
UL49, UL50, and most likely the
intervening UL49.5. Since TCC 4.2E1 and 2.3 react
with RS1G31 and DX32, but not with RP2 (Fig. 1), recognition of
UL49, UL49.5, or UL50 is most
likely.
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FIG. 1.
(Top) Organization of the HSV genome in the region of
map units 0.67 to 0.73. Boundaries are approximate. HSV-1 × HSV-2
IRV are also shown. HSV-2 DNA is indicated by a solid line, HSV-1 DNA
by a dashed line, and indeterminate regions by multiple lines. The
HSV-2 BamHI w fragment used for expression cloning is also
shown. ORF, open reading frame. (Bottom) Proliferative responses of TCC
to the indicated IRV. Data are cpm, expressing
[3H]thymidine incorporation compared to that in the
medium, which was less than 500 cpm in each case.
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FIG. 2.
Determination of the HSV viral phenotype of the
UL49 gene product (VP22) of IRV DX32. Lysates of
mock-infected cells and cells infected with the indicated viral strains
were separated by SDS-polyacrylamide gel electrophoresis, blotted, and
probed with VP22-specific MAb. The molecular weights (in thousands) of
marker proteins are shown at the right.
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Expression cloning to determine T-cell antigens.
The
BamHI w fragment of HSV-2 was selected for expression
cloning, since it contains the UL49 and UL49.5
coding sequences and most of the UL50 coding sequence
(6, 11, 24). Seventy to ninety percent of random colonies
contained an insert; all were of viral origin. The most active
libraries (Table 1) for each TCC (pUEX1
for TCC 4.2E1 and pUEX3 for TCC 2.3) were selected, and an individual
reactive bacterial clone was detected by sequential testing of pools
and individual colonies (Table 2). Clone
1.1.3 encodes a fusion protein eliciting proliferation by TCC 4.2E1. This clone contains a backwards 80-bp SmaI fragment of
UL49, a 262-bp SmaI fragment of HSV-2
UL49 DNA predicted to encode amino acids 105 to 190, forward and in frame with regard to -galactosidase, and a 246-bp
SmaI fragment of UL49 forward but out of frame
due to a deletion of a single C residue at the 262-bp SmaI
fragment-242-bp SmaI fragment junction. Clone 3.19 contained a 583-bp SmaI fragment encoding amino acids 118 to
312 of UL50, followed by backwards 80- and 96-bp
SmaI fragments of UL49.
Identification of T-cell antigens was confirmed by targeted subcloning
and overlapping peptides. The 262-bp
SmaI fragment
of
U
L49 of HSV-2 encoding amino acids 105 to 190 was subcloned
into pUEX3 to yield plasmid 49.262.12. This protein stimulated
TCC 4.2E1 (Table
2). Only peptide 105-126 of VP22 of HSV-2
(GGPVGAGGRSHAPPARTPKMTR)
was stimulatory (Fig.
3). DNA fragments encoding
U
L50 118-312
and 118-250 were subcloned into pUEX3. Fusion
proteins expressing
these fragments were active (Table
2).
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FIG. 3.
Proliferative responses of VP22-specific TCC to peptide
epitopes in VP22 of HSV-2. APC were autologous EBV-LCL (for TCC
4.2E1) or autologous PBMC (for other TCC). Antigens included
-galactosidase and fusion proteins used at 10 µg/ml and peptides
used at 3 µM (for TCC 4.2E1) or 1 µM (for other TCC). Data are
cpm, expressing [3H]thymidine incorporation compared
to that in the medium, which was less than 500 cpm in each case.
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Evaluation of random colonies from full-length HSV-2 DNA
libraries showed that 80 to 100% contained plasmids with an
insert;
80 to 100% of inserts were of viral origin. For both TCC
ESL4.9
and ESL2.20, only the pUEX3 protein library elicited
lymphoproliferation
(Table
1). Since the libraries were more complex
than those made
from the
BamHI w fragment, 2,000 to 3,000 bacterial transformants
were screened by a combinatorial method. In
preliminary experiments,
heat-killed, washed bacteria were found to
substitute for inclusion
body preparations of protein in
lymphoproliferation assays at
the pool (5 to 12 bacterial clones) and
final-assay stages (data
not shown).
Sequencing of plasmids in positive bacteria showed that TCC
ESL4.9 recognized a 44-amino-acid fragment of U
L49
gene product
VP22 (amino acids 177 to 220), while TCC ESL2.20
recognized a
34-amino-acid fragment of the U
L21 gene
product (amino acids 148
to 181) (Table
2). In both cases, single
AluI fragments of HSV-2
DNA were inserted in frame and
forward. Peptide mapping revealed
that amino acids 187 to 206 (Fig.
3)
stimulated TCC ESL4.9.
Fusion proteins as probes of bulk lesion-infiltrating T cells.
Newly discovered T-cell antigens were added to the panel of HSV
antigens used to probe bulk cultures of lesion-infiltrating T cells.
The first available specimens in our ongoing studies (19) were a set of four biopsy specimens (2 mm each)
obtained from a day-5 (virus culture positive) buttock lesion due to a recurrence of HSV-2 in patient 1 (17). All four biopsy
specimens showed reactivity with VP22 105-190 but not with
-galactosidase, glycoprotein B or D, or VP16 (data not shown).
TCC were derived after the original bulk culture was
restimulated for one cycle with VP22 105-190 fusion protein. The
proliferative responses of TCC 1.L3D5.10.8 (Fig. 3) to VP22 105-190 and
constituent peptides document a third T-cell epitope in VP22,
contained within amino acids 125 to 146.
Definition of additional T-cell epitopes in tegument protein
VP16.
We previously found three epitopes within VP16 (Table
3), all HSV-2 type specific
(14), and detected proliferative responses to full-length
VP16 in bulk cultures of genital HSV-2 lesion-infiltrating lymphocytes
from four of seven (57%) patients (19). We sought additional peptide epitopes within VP16 by two strategies. The first strategy was to screen panels of clones recovered from lesion vesicle fluid for reactivity with recombinant VP16 of HSV-2, followed by epitope mapping with peptides. Peptides containing amino acids 185 to 197 and the overlapping pair 209-221 and 213-225 were
stimulatory for TCC RH.13 and KM.7, respectively (Table 3). All other
VP16 peptides were negative (<500 cpm [data not shown]). The second strategy involved use of PBMC as the starting material and secondary in
vitro stimulation with recombinant baculovirus-derived VP16. Clones
(BM.17 and SB.17) from two individuals recognized the same peptide
(amino acids 437 to 449), as well as a -galactosidase-VP16 fusion
protein and whole virus. All three newly defined VP16 epitopes were
type common, shared by HSV-1 and HSV-2 whole-virus preparations, as
expected from sequence data (6).
HLA restriction.
The HLA restriction of the TCC-recognizing
antigens encoded near 0.7 map units was determined in detail.
Proliferation of TCC 4.2E1, specific for VP22 105-126, is inhibited
84% by an anti-DP MAb but less than 20% by an anti-DR or anti-DQ MAb.
TCC 4.2E1 is from a DPB1*2001/DPB1*0402 heterozygous donor. Allogeneic
EBV-LCL bearing DPB1*2001 but not DPB1*0402 present antigen (Table
4), establishing restriction by
DPB1*2001. Proliferation of TCC 2.3, specific for UL50, was
inhibited by an anti-DR MAb but not by an anti-DP or anti-DQ MAb. This
clone is from a DRB1*0301/BRB1*0701 heterozygous donor.
Allogeneic PBMC from a DRB1*0301 donor presented antigen,
consistent with binding of antigenic peptide to this allele.
However, presentation by one of the linked DR gene products DRw52
and DRw53 cannot be ruled out. Additional HLA restriction studies
are summarized in Table 5.
The HLA restriction of TCC BM.17 was studied in detail, since the
antigenic peptide, VP16 437-449, overlaps two previously
described (
18,
21) VP16 epitopes (Table
3).
Proliferation
of TCC BM.17 and the similar clone SB.17 was
inhibited 90% by
an anti-DQ MAb but less than 25% by an anti-DR or
anti-DP MAb.
Donors 8 and 9 are heterozygous for HLA
DQB1*0201/0501. At high
concentrations of peptide, both DQB1*0201
and DQB1*0501 homozygous
EBV-LCL appeared to present antigen to TCC
BM.17. However, DQB1*0501
homozygous cells presented peptide much
more efficiently than
DQB1*0201 homozygous cells (Fig.
4). Thus, three different but
overlapping epitopes in VP16 431-449 are presented
by HLA DQB1*0302,
DQB1*0201, and DQB1*0501.
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FIG. 4.
HLA restriction element for TCC BM.17 response to
peptide 437-449 of VP16 of HSV-2. Proliferative responses are plotted
against concentrations of viral peptide. APC are EBV-LCL that are
either autologous (solid circles), homozygous for HLA DQB1*0501 (open
inverted triangles), or homozygous for HLA DQB1*0201 (solid squares).
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CTL activity of tegument-specific CD4 TCC.
Cytotoxic
activities of the CD4 TCC with newly and previously identified
specificities were tested by using EBV-LCL target cells (Table
5). All clones tested displayed cytolytic
activity towards peptide-loaded target cells. Cytolytic activity
against target cells infected with HSV-2 showed greater
variability. VP22-specific TCC 4.2E1 was active, while VP22-specific
TCC from other donors were not. Among the seven VP16-specific TCC
tested, six displayed greater than 10% cytotoxicity towards
HSV-2-infected target cells. The single UL21- and
UL50-specific TCC were not active against virus-infected
target cells.
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TABLE 5.
Cytolytic activity of lesion-derived, tegument-specific
CD4 TCC, with summary of fine specificity and HLA restriction
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DISCUSSION |
HSV-specific T cells selectively infiltrate recurrent genital
HSV-2 lesions (16). Local CTL activity, with CD4- and
CD8-mediated components, is correlated with viral clearance
(19). The antigens recognized by local HSV-specific T cells
are diverse and in many cases unknown (17). In this report,
we document recognition of tegument proteins VP22 and UL21
and the viral dUTPase, and we extend our previous observations on
tegument protein VP16.
HSV has several characteristics which facilitate the expression cloning
system described in this report. Genomic double-stranded DNA can be
used directly, since introns are rare in the HSV genome. The same HSV-2
strain, HG52 (9), was used to screen candidate lesion-derived TCC and make protein libraries. The relatively low
degree of strain variability (28) between HSV-2 strains in the donors and HG52 might rarely lead to omission of an
epitope(s) recognized in vivo; application to viruses with more
strain variation would benefit from the use of autologous isolates.
Notably, reactivity with VP22 was detected in two independent
expression cloning experiments with lesion-infiltrating TCC from two
donors. VP22 reactivity was also detected during screening of the first
available set of bulk lesion-infiltrating lymphocyte cultures. We plan
assessment of the reactivity of bulk and cloned T cells from the
herpetic lesions of additional subjects after expression of full-length
antigens. Thus far, 10 additional clones from three patients have been
negative with fragments of UL49, UL21, and
UL50 discussed in this report; assessment of the relative immunodominance of tegument-specific responses in comparison to HSV
glycoproteins is not possible at this time.
Tegument antigens may be suitable targets for lesion-infiltrating CD4 T
cells because of their abundance. VP16 and VP22 are present in large
amounts: on the order of 1.6 × 103 molecules of VP16
(45) and 2.5 × 103 to 2.8 × 103 molecules of VP22 (23) are incorporated into
each virion in HSV-1. Less information is available for
UL21 (2, 3). The viral dUTPase is the first
nonvirion component documented to be a target of the HSV-specific CD4
T-cell response; no information is available concerning its relative
abundance. This enzyme, like VP16 and VP22, localizes to the nuclei of
cells infected with HSV-2 (but not HSV-1) (40). Antigen
presentation in vivo may occur after endogenous synthesis of dUTPase in
infected cells or scavenging of dUTPase antigen from
infected-cell debris. Lysis of HSV-infected cells by
dUTPase-specific TCC 4.2E1 indicates that, at least in vitro,
presentation of endogenous antigen can occur.
Immune responses to VP22 may be an obstacle to its use for delivery of
exogenous materials (30) in vivo. Conversely, since polypeptides expressed as C-terminal fusions to VP22 can be
cotransported into cells, expression of proteins as VP22 fusions may be
of interest as a type of adjuvant preparation. This hypothesis can
be tested by expression of heterologous epitopes in VP22. VP16 and
VP22 of HSV-1 are strongly, noncovalently associated in infected
cells as shown by coimmunoprecipitation. These proteins colocalize in the perinuclear areas of cells (10, 11). Possibly, this
association plays a role in stimulating the apparently high level of
CD4 T-cell response to VP16.
All of the lesion-derived tegument-specific CD4 TCC studied had
cytolytic potential against maximally sensitized (peptide-loaded) target cells. Why do only some clones kill virus-infected cells? Possibly, some epitopes are not presented in infected APC, and some
may be processed at very low levels, below a threshold needed to
trigger a cytolytic response. Thus far, with our small number of
clones, we have not observed a consistent relationship between antigenic specificity or HLA restricting locus and sensitization, by
viral infection, to lysis by CD4 T cells.
In summary, expression cloning has allowed the discovery of novel HSV
T-cell antigens. The in situ enrichment of antigen-specific CD4 T cells
in lesions allows study of the antigenic repertoire unbiased by
secondary in vitro stimulation with antigen. The favorable characteristics of the HSV genome allow direct use of libraries of
whole-virus DNA. In the future, a more complete description of the
diversity and targets of HSV-specific T cells and correlation of these
factors with the duration, clinical severity, or complications of HSV
infections, such as HSV keratitis, may be assisted by application of the methods in this report. Tegument proteins are candidates, together with membrane glycoproteins, for evaluation as possible HSV
vaccines in humans.
| |
ACKNOWLEDGMENTS |
This study was supported by NIH grants AI34616 and CA70017 (to
D.M.K.).
HLA typing was performed by John Hansen and Effie Petersdorf.
HSV-1 × HSV-2 IRV were graciously provided by Bernard Roizman (RS1G31), Howard Marsden (DX32), and Steve Triezenberg (RP-2). The
HSV-2 HG52 BglII i fragment was provided by Steve
Triezenberg and originally by Chris Preston. MAb p43, specific for
VP22, was provided by David Meredith. John C. Hutton provided
pUEX vectors, primer sequences, and valuable advice. Peptides and
recombinant VP16, gB2, and gD2 were kindly provided by Rae L. Burke and
Michael Tigges, Chiron Corporation. Lawrence Corey provided invaluable support and advice over several years. We thank Jeffrey Vieira for
valuable advice and Mike Remington, Gail R. Barnum, and Mary Shaughnessy for assistance with specimen collection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fred Hutchinson
Cancer Research Center, Room D3-100, 1100 Fairview Ave. North, P.O. Box
19024, Seattle, WA 98109. Phone: (206) 667-6807. Fax: (206) 667-6707. E-mail: viralimm{at}u.washington.edu.
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Abstract
Introduction
