Journal
of General Virology (1998), 79, 353–361. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Herpes simplex virus type 1 glycoproteins gB, gC and gD are
major targets for CD4 T-lymphocyte cytotoxicity in HLA-DR
expressing human epidermal keratinocytes
Zorka Mikloska and Anthony L. Cunningham
Centre for Virus Research, Westmead Institutes for Health Research, Westmead Hospital, Westmead, and University of Sydney,
NSW 2145, Australia
T lymphocytes are the main mediators of the
protective immune response in recurrent herpes
simplex. Early in the development of recurrent
lesions, macrophages and CD4 T lymphocytes predominate in the mononuclear infiltrate surrounding
infected epidermal cells. Human epidermal keratinocytes allow herpes simplex virus type 1 (HSV-1)
replication and human leukocyte antigen (HLA)-DR
is strongly expressed in vivo. In vitro, their pretreatment with IFN-γ induced HLA-DR expression
and partially reversed major histocompatibility
complex class I down-regulation by the virus. Mononuclear cell cytotoxicity for these cells was mediated
predominantly by CD4 and also by CD8 T cells. Late
HSV-1 proteins were the major targets for CD4 CTL,
while CD8 CTL predominantly targeted early HSV-1
proteins. Here it is shown that both mononuclear
Introduction
The immune control of recurrences of human herpes
simplex infection appears to be mainly mediated by T
lymphocytes and macrophages. CD4 lymphocytes and macrophages are the predominant infiltrating cells in the early stages
of the lesions as determined by immunohistology and direct
cloning of T lymphocytes from the lesions (Cunningham et al.,
1985 ; Koelle et al., 1994 a, b). B lymphocytes are scarce in the
mononuclear infiltrates in recurrent herpes lesions and no
correlation has been established between the herpes simplex
virus (HSV) antibody titres or neutralizing antibody titres and
frequency of recurrence (Reeves et al., 1981). However, high
levels of pre-existing neutralizing antibody may still play a role
in the prevention of spread of HSV, especially in the prevention
of viraemia.
The persistence of herpes lesions in AIDS and transplant
Author for correspondence : A. L. Cunningham.
Fax ­61 2 9845 8300. e-mail tonyc!westmed.wh.su.edu.au
and CD4 CTL consistently recognized the major
HSV-1 glycoproteins, gB, gC, gD and gH, using IFNγ-pretreated keratinocytes infected with vaccinia
virus–HSV glycoprotein recombinants (VvgB, VvgC,
VvgD or VvgH). CD4 cytotoxicity was highest for
VvgD-infected keratinocytes, followed by VvgB or
VvgC and then VvgH in seven patients. CD4 CTL
from two of 13 patients also recognized an epitope
in the HSV tegument protein VP16, demonstrated
by comparing cytotoxicity for the partial deletion
mutants RP3 or RP4 and the parental RP1 HSV
strain. In summary, the major HSV glycoproteins gB,
gC and gD were consistently the major targets for
CD4 CTL in VvgB-, VvgC-, VvgD- and VvgH-infected,
IFN-γ-pretreated human epidermal keratinocytes in
vitro.
patients where the immune defect is predominantly, but not
solely, in CD4 T lymphocytes and macrophages also suggests
a major role for these cells (Siegal et al., 1981 ; Fauci, 1988). The
presence of high levels of IFN-γ in vesicle fluid and the
correlation of titres of IFN-γ in vesicle fluid or that secreted by
blood T cells with the frequency of recurrence also supported
the importance of CD4 cells in the control of recurrent herpes
simplex lesions (Cunningham & Merigan, 1983, 1984 ; Torseth
& Merigan, 1986). High titres of IFN-α and IFN-β in vesical
fluid, probably released from epidermal cells and}or macrophages, also suggest an important role of these cytokines in the
prevention of spread, especially in the early stages before restimulation of immune T cells (Overall et al., 1981). Another
function of IFN-γ within the lesions is the induction of major
histocompatibility complex (MHC) class II antigens [human
leukocyte antigens (HLA)-DR, not -DQ] on the surface of
epidermal cells within the first 2 days of the appearance of
lesions (Cunningham et al., 1985 ; Cunningham & Noble,
1989).
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Z. Mikloska and A. L. Cunningham
Hence, the early infiltrating CD4 T lymphocytes may exert
immune control through the secretion of cytokines such as
IFN-γ or possibly through CD4 T-lymphocyte cytotoxicity.
Although the latter is controversial, there are two reasons to
consider that CD4 T-lymphocyte cytotoxicity may be important in recurrent herpes simplex lesions. The first is the
timing of events within the lesions. The initial infiltration of
CD4 T lymphocytes correlates with the reduction of virus
titres within vesicle fluid, whereas on average the later
infiltration of CD8 T lymphocytes occurs after 2 days when
vesicle fluid titres of virus have already declined (Cunningham
et al., 1985 ; Spruance et al., 1977). Secondly, although it has
been known for many years that human CD8 T lymphocytes
exert very weak cytotoxicity against HSV-infected fibroblasts
(Posavad & Rosenthal, 1992 ; Posavad et al., 1993 ; Koelle et al.,
1993), only recently has the mechanism been established. HSV
proteins, including ICP47, complex with the translocator
protein associated with antigen presentation (TAP) preventing
the reconstitution of MHC I with antigenic peptides and
leading to trapping of MHC class I in the endoplasmic
reticulum (York et al., 1994 ; Fruh et al., 1995 ; Hill et al., 1995).
However, we have recently demonstrated that preincubation
of keratinocytes with IFN-γ partially reverses this effect on
HSV-infected keratinocytes and that MHC class II antigen
expression was unaffected by HSV infection, enabling interaction between CD4 T lymphocytes and epidermal cells
(Mikloska et al., 1996). Previously, we have demonstrated that
T lymphocytes re-stimulated in vitro from the blood of HSVimmune patients are cytotoxic for HSV-infected keratinocytes
which have been stimulated to express HLA-DR by IFN-γ
pretreatment (Cunningham & Noble, 1989 ; Mikloska et al.,
1996). CD4 T-lymphocyte cytotoxicity was always greater
than CD8 T-lymphocyte cytotoxicity despite the partial
restoration of MHC class I antigen expression on the surface of
these cells. The predominant targets of CD4 T-lymphocyte
cytotoxicity were late viral proteins (Mikloska et al., 1996).
Here we have further defined the targets for CD4 Tlymphocyte cytotoxicity using a battery of vaccinia virus–HSV
glycoprotein recombinants expressing the major HSV glycoproteins, gB, gC, gD and gH. We have also examined
cytotoxicity for one of the two major tegument proteins,
VP16, which is also a late protein (Ward & Roizman, 1994).
These studies are highly relevant to the current trials of
recombinant glycoproteins as candidate HSV vaccines for
primary prophylaxis and therapeutic efficacy.
Methods
+ Patients and epidermal cultures. The tissues were obtained
from HSV-1 seropositive patients undergoing split skin grafting for
surgical procedures only after informed consent and under approval from
the Western Sydney Area Health Service Research and Ethics Committee.
Immediately after the operation, skin specimens were aseptically
transferred into minimum essential medium (MEM) (Gibco) containing
Earle’s salt and other ingredients as described previously (Cunningham &
DFE
Noble, 1989 ; Mikloska et al., 1996) and epidermis was isolated as
described. One to two months after recovery 60–100 ml of heparinized
blood was drawn for isolation of peripheral blood mononuclear cells
(PBMC).
The skin cultures were grown in growth medium (MEM) as described
previously (Cunningham & Noble, 1989). Viability was determined using
trypan blue exclusion and the viable cells were subcultured in 96-well flatbottom plates (Nunc) at a density of 10% cells per well in growth medium
for 18 h at 37 °C in 5 % CO prior to the addition of IFN-γ and}or
#
infection with HSV-1. The viruses used were the RP1 KOS strain, RP2,
RP3, or RP4 VP16 deletion mutant viruses of HSV KOS, wild-type
vaccinia virus or vaccinia virus–HSV glycoprotein recombinants. For
immunofluorescence experiments the explants or MRC-5 were grown in
four-well tissue culture chambers (Nunc) to confluency.
+ HSV, HSV antigen and vaccinia virus–HSV glycoprotein
recombinants. The wild-type HSV strains used in all cytotoxicity
experiments were the F strain and a low passage clinical isolate of HSV1 (WM2) (Cunningham & Noble, 1989). The RP1 KOS strain (which is
HSV-1 KOS, with a BamHI site in the 3« region of the VP16 sequence)
and the derived VP16 mutants RP2 (RP1, with the VP16 gene from HSV2), RP3 (RP1, with deletion of amino acids 456–490) and RP4 (RP1, with
deletion of amino acids 413–490) were all kindly provided by S.
Triezenberg (Michigan State University, USA) and constructed in his
laboratory by Rath Pichyangkura (Cress & Triezenberg, 1990 ;
Weinheimer et al., 1992). These strains were used to infect human
keratinocyte cultures. HSV antigen was prepared by UV inactivation of
supernatants of HSV-1-infected HEp-2 cells and therefore includes both
viral (structural) and non-structural (infectious) cellular proteins. Mock
(control) antigen was produced by omitting HSV infection.
WR wild-strain vaccinia virus was used as a control and was kindly
provided by I. Ramshaw (Australian National University, Canberra).
Vaccinia recombinants VvgB (expressing HSV glycoprotein B) (Cantin et
al., 1987), VvgC5 (Martin et al., 1993) and VvgD13 were kindly donated
by B. Rouse (University of Tennessee, Knoxville, USA) and VvgH
(Forrester et al., 1991) by A. Minson (University of Cambridge, UK) who
also provided monoclonal antibody to gH. VvgC and VvgD were under
the control of early promoters, VvgB was under late promoter control
and VvgH was under early and late promoter control. WR strain vaccinia
virus and vaccinia virus–HSV recombinants were grown in CV-1 cells.
The same cells were used for quantification of vaccinia virus titre by the
plaque assay.
+ Infection of keratinocytes with viruses and quantification of
HSV protein expression by immunofluorescence. Keratinocytes
and HEF monolayers were infected with the WM2 clinical strain or the
RP1 strain or the RP2, RP3 or RP4 VP16 mutant viruses at 10 p.f.u. per
cell of HSV-1, also with 15 p.f.u. per cell of vaccinia virus–HSV
glycoprotein recombinants or wild-strain vaccinia virus in 96-well tissue
culture plates (for cytotoxicity experiments) or four-well tissue culture
chambers (for immunofluorescence staining). For cytotoxicity experiments cells were incubated with the virus for 1 h at 37 °C and then the
growth medium was added. For immunofluorescence staining, keratinocytes infected with HSV VP16 deletion mutants or RP1 control virus for
24 h were fixed with methanol and incubated for 1 h with monoclonal
antibody to gD1 (Cymbus). After double washing, cells were labelled
with FITC-conjugated F(ab«) rabbit anti-mouse immunoglobulin
#
(Dakopatts). The proportion and the intensity of gD staining within cells
were estimated (see below). The expression of gB, gC, gD and gH by
keratinocytes infected with vaccinia virus–HSV glycoprotein recombinants was checked in a kinetic study. Keratinocytes grown in tissue culture
chambers were pretreated with 100 IU}ml IFN-γ for 4 days, infected with
VvgB, VvgC, VvgD or VvgH at an m.o.i. of 15 p.f.u. per cell for 1 h,
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HSV-1 targets for human CD4 cytotoxicity
washed as for a cytotoxicity assay and fixed at 0, 6, 8, 10, 12, 14, 18 and
24 h after infection. The slides were incubated with antibody for FITClabelled anti-gC1 (Syva Microtrak), unlabelled anti-gB1 (Chemicon),
unlabelled anti-gD1 (Cymbus) and unlabelled anti-gH at saturating
concentrations. Rabbit FITC-labelled F(ab«) to mouse immunoglobulin
#
was then incubated with cells for a further 30 min. Controls using
irrelevant monoclonal antibodies and uninfected keratinocytes were
included in each experiment. One hundred cells were counted on each of
10 randomly chosen fields of the infected monolayer. The number of
positive (infected) cells was recorded and the mean and the SE for control
cells and IFN-γ-pretreated cells were calculated. The overall intensity of
staining was estimated semi-quantitatively on a scale of 1 to 4 in
comparison with keratinocytes infected with HSV at an m.o.i. of 10 p.f.u.
per cell (4­ intensity).
+ PBMC and lymphocyte subset preparation. Approximately 1
month after initiation of the keratinocyte cultures, autologous blood was
drawn. PBMC were isolated from heparinized blood samples over
Ficoll–Hypaque density gradients. After 6 days of incubation with HSV
antigen, PBMC were depleted from CD4 and natural killer (NK) cell
(PBMC-CD8 cells) and CD4 T-lymphocyte (PBMC-CD8-NK cells)
subsets using murine monoclonal antibodies and complement-dependent
cytotoxicity as previously described (Cunningham & Noble, 1989). The
number of CD4 lymphocytes increased by 30–40 % due to the 6 day
stimulation with HSV antigens whereas CD8 cells remained the same or
declined slightly. Monoclonal antibodies to CD4 (anti-human Leu 3a and
3b), CD8 (anti-human Leu 2a) and NK cells (anti-human Leu 16) were
obtained from Becton Dickinson. Residual contaminating cells in samples
of PBMC and lymphocyte subsets were quantified by indirect immunofluorescence with OKT4 and OKT8 (Orthomune), respectively, and flow
cytometry as described previously (Cunningham & Noble, 1989) and
found to be ! 5 % (data not shown).
+ Cytotoxicity assays. Keratinocytes were subcultured in 96-well
flat-bottom plates (as described above) and pretreated for 4 days with
growth medium or growth medium containing 100 IU}ml of recombinant IFN-γ (Boehringer Mannheim). The cells were infected with
HSV-1 as described previously (Cunningham & Noble, 1989 ; Mikloska et
al., 1996), using RP1, RP2, RP3 or RP4 mutant viruses at 10 p.f.u. per cell,
or with vaccinia virus control or vaccinia virus–HSV glycoprotein
recombinants at an m.o.i. of 15 p.f.u. per cell for 1 h at 37 °C, washed
twice with Hank’s balanced salt solution and incubated with 5 µCi per
well of sterile &"Cr sodium solution (Amersham) for 90 min. In control
wells virus was omitted. To determine the dependence of lysis on HLADR expression after infection, some keratinocytes were incubated with
5 µg}ml of anti HLA-DR antibody (Becton Dickinson) for 2 h. After
washing and subsequent incubation with 5 µCi per well of sterile &"Cr for
90 min they were mixed with CD4 lymphocytes in an effector to target
ratio of 25 : 1, 50 : 1, 80 : 1 and 100 : 1. Anti-HLA-DR antibody was
maintained at 5 µg}ml for the duration of the 18 h incubation.
Mononuclear cells were stimulated with control or HSV-1 antigen for
6 days. On the day of experiment, cells were depleted of CD8 and}or
CD16 cell subsets as described above, and were incubated (with targets
at an effector to target ratio of 80 : 1) for 18 h, which was found to be
optimal (for HSV-1, RP1–RP4 and also vaccinia virus control}vaccinia
virus–HSV recombinants) in past and further preliminary experiments
(see Results and Table 3) (Cunningham & Noble, 1989 ; Mikloska et al.,
1996).
Supernatant radioactivity was quantified in a gamma-radiation
counter (Beckmann) and the percentage of specific cytotoxic activity was
determined by the following equation : % specific &"Cr release ¯ [(mean
experimental c.p.m.®mean spontaneous c.p.m.)}(mean maximal
c.p.m.®mean spontaneous c.p.m.)]¬100.
Some of the cytotoxicity results were expressed as the mean net
specific lysis : mean net specific lysis ¯ (mean specific lysis for vaccinia
virus–HSV glycoprotein recombinant)®(mean specific lysis for vaccinia
virus control), where mean refers to the means of replicate experiments.
+ Statistics. Standard errors of experimental c.p.m. (triplicates and
quadruplicates) were usually less than 10 %. Differences between the
percentage of specific &"Cr release obtained with different treatments
were assessed for statistical significance by Student’s t-test with
modification of the degrees of freedom to allow for unequal variances
(Cunningham & Noble,1989).
Results
Expression of HSV glycoproteins in keratinocytes
infected with vaccinia virus–HSV glycoprotein
recombinants
The proportion and the intensity of gB, gC, gD and gH
glycoprotein staining in infected cells at an m.o.i. of 15 p.f.u.
per cell was examined by direct immunofluorescence at 0, 6, 8,
10, 12, 14, 18 and 24 h after infection, using murine monoclonal
antibodies to each of the glycoproteins (Table 1). A high
proportion of keratinocytes had high-intensity cytoplasmic
and membrane glycoprotein expression after infection with the
same m.o.i. and for the same time as for the cytotoxicity assay
(Table 1). However, the proportion of cells expressing gH was
consistently lower (significant in two out of three experiments)
than for the other glycoproteins despite the use of the same
m.o.i. The proportion of cells which stained at 18 h when
Table 1. Expression of HSV-1 glycoproteins gB, gC, gD
and gH in IFN-γ-pretreated keratinocytes 18 h after
infection with recombinant vaccinia virus
Glycoprotein
gB
gC
gD
gH
% Positive cells
(³SE)
Intensity
83³12
95³6
87³11
61³22
3­
3­
3­
3­
Table 2. Expression of gD1 in IFN-γ-pretreated
keratinocytes 24 h after infection with the RP3 VP16
partial deletion mutant virus in comparison with the
parental strain KOS
Virus strain
KOS
RP3
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% Positive
cells (³SE)
Intensity
81³12
75³7
4­
4­
DFF
Z. Mikloska and A. L. Cunningham
infected with the HSV-1 control was 90³3 %. Negative and
time 0 controls showed no staining.
As shown in Table 2, the proportion of keratinocytes
expressing HSV gD1 in cells infected with the VP16 mutants
RP3 or with the RP1 KOS control was similar. Similar results
were also obtained after infection of keratinocytes with RP2
and RP4 mutants. There was also no difference in the (very
high) intensity of staining.
Table 3. CD4 T-cell cytotoxicity against IFN-γ-pretreated
keratinocytes infected with vaccinia virus–HSV
glycoprotein recombinant : determination of optimal
effector to target ratio
Results are expressed as % specific lysis of keratinocytes (mean³SE).
Effector to target ratio
Target
Comparison of PBMC, CD4 and NK and CD4 T cells as
effectors in cytotoxicity for keratinocytes infected with
vaccinia virus–HSV glycoprotein recombinants or
VP16 deletion mutants
Cytotoxicity for vaccinia virus–HSV glycoprotein recombinants was compared between PBMC, CD4 and NK and CD4
T effectors in four patients. These comparisons were carried
out in one of the patients on six different occasions. In addition,
variability in recognition of different HSV glycoproteins or
VP16 was examined by using keratinocyte targets and
mononuclear cell effectors from a number of patients : five
different patients for PBMC, six for the CD4 and NK cell
subset and 20 for the CD4 T-cell subset (seven for glycoproteins and 13 for the VP16 protein target). Experiments were
usually repeated on two separate occasions for each patient.
In several preliminary experiments the optimal effector to
target ratio and incubation time were examined with the
vaccinia virus–HSV glycoprotein recombinants and the VP16
deletion mutants. The optimal effector to target ratio, probably
determined by target cell type and size, was 80 : 1 (Table 3) and
incubation time was 18 h (data not shown) as previously
reported (Cunningham & Noble, 1989).
In four patients, mononuclear cell cytotoxicity for gB, gC,
gD and gH was slightly greater (10–12 % for gB, gC, gD and
5 % for gH) than for CD4 and NK (data not shown) or CD4 Tcell cytotoxicity (Fig. 1). However, the latter two were almost
identical (Fig. 1 and Table 3) indicating the reproducibility of
the experiments and the lack of recognition of these glycoproteins by NK cells, as shown in a previous report
(Cunningham & Noble, 1989). Cytotoxicity for negative and
positive controls (vaccinia virus and HSV-1) was not significantly different for mononuclear cells, CD4 and NK and CD4
cells.
PBMC cytotoxicity for IFN-γ-pretreated and vaccinia
virus–HSV recombinant-infected keratinocytes
In four of five patients, mononuclear cells lysed IFN-γpretreated and VvgD-infected keratinocytes (specific lysis of
61 %) to a significantly greater extent than VvgB- (45 %)
followed by VvgC- (29 %) and VvgH- (18 %) infected cells (Fig.
1). In one patient, lysis of VvgC-infected targets was the
greatest (68 %) in comparison with VvgD (46 %), VvgB (38 %)
and VvgH (22 %), but this hierarchy was not maintained for
CD4 and CD4 and NK cytotoxicity.
DFG
HSV-1
Vaccinia control
VvgB
VvgC
VvgD
VvgH
25 : 1
50 : 1
80 : 1
100 : 1
45³0±6
12³1±3
37³0±6
34³0±9
42³0±8
20³0±8
52³0±4
15³0±9
42³0±9
35³0±8
46³1±4
21³0±9
72³1±2
14³0±7
44³0±8
39³0±7
61³0±9
24³0±3
70³0±9
11³0±9
40³0±7
37³0±9
60³0±5
21³0±4
(a)
(b)
Fig. 1. Comparison of PBMC (a) and CD4 (b) cell cytotoxicity for IFN-γpretreated and vaccinia virus–HSV glycoprotein recombinant-infected
keratinocytes in the same person. Histograms represent mean of four
replicates³SE. Similar results were obtained in three other subjects.
Effector to target ratio was 80 : 1. VvgB, vaccinia virus–HSV glycoprotein B
recombinant-infected targets ; VvgC, vaccinia virus–HSV glycoprotein C
recombinant-infected targets ; VvgD, vaccinia virus–HSV glycoprotein D
recombinant-infected targets ; VvgH, vaccinia virus–HSV glycoprotein H
recombinant-infected targets.
Reproducibility was tested by repeated experiments (six
times) with the same patient’s PBMC as effectors and
keratinocytes as targets. No significant differences in the above
hierarchy of results were observed on six separate occasions
(P ! 0±05).
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HSV-1 targets for human CD4 cytotoxicity
Table 4. CD4 cytotoxicity for IFN-γ-stimulated keratinocytes infected with vaccinia
virus–HSV glycoprotein recombinants (mean net specific lysis)
Effector to target ratio was 80 : 1. Mean net specific lysis ¯ (mean specific lysis for vaccinia virus–HSV
glycoprotein recombinant)®(mean specific lysis for vaccinia control). Means are derived from the means of
replicate experiments for each of 13 patients.
CD4 and NK cells
Target
VvgB
VvgC
VvgD
VvgH
HSV-1 control
Vaccinia control
CD4 T cells
Mean (%)
Range (%)
Mean (%)
Range (%)
41
35
53
30
76
18
37–45
32–38
49–56
27–33
74–79
14–20
38
32
48
26
65
18
32–43
28–36
45–54
22–29
61–67
15–19
(a)
(b)
Fig. 3. Effect of anti HLA-DR antibody on CD4 lymphocyte cytotoxicity for
HSV-infected keratinocytes. Anti HLA-DR antibody was incubated with
HSV-infected, IFN-γ-stimulated keratinocytes and mixed with CD4 effector
T cells at a ratio of 80 : 1 as described in Methods.
Fig. 2. Comparison of CD4 cell cytotoxicity for IFN-γ-pretreated and
vaccinia virus–HSV glycoprotein recombinant-infected keratinocytes. (a)
Results from one patient representative of five of seven patients and (b)
results from one patients representative of the other two patients. Effector
to target ratio was 80 : 1. VvgB, vaccinia virus–HSV glycoprotein B
recombinant-infected targets ; VvgC, vaccinia virus–HSV glycoprotein C
recombinant-infected targets ; VvgD, vaccinia virus–HSV glycoprotein D
recombinant-infected targets ; VvgH, vaccinia virus–HSV glycoprotein H
recombinant-infected targets.
CD4 and NK and CD4 T-cell cytotoxicity for IFN-γpretreated and vaccinia virus–HSV recombinantinfected keratinocytes
There was little difference between CD4 and NK cell and
CD4 T-cell cytotoxicity, hence they will be considered
together.
With both CD4 and CD4 and NK populations, cytotoxicity
for keratinocytes infected with VvgD1 was usually the highest
(mean net specific lysis 53 % for CD4 and NK ; 48 % for CD4 Tcell cytotoxicity) (Table 4) of all recombinants tested under
conditions of equal m.o.i. and similar HSV glycoprotein
expression (see above). In seven out of nine patients, including
five out of seven with CD4 lymphocytes as effectors, there was
a hierarchy in cytotoxicity for gD " gB " gC " gH at all
effector to target ratios (Tables 3 and 4).
After VvgD1, the mean net specific lysis and % specific
lysis for keratinocytes infected with VvgB was next highest
(mean 41 % for CD4 and NK cells, 38 % for CD4 T cells)
followed by VvgC (mean 35 % for CD4 and NK cells, 32 % for
CD4 T cells), and VvgH (mean 30 % for CD4 and NK cells,
26 % for CD4 T cells) (Table 4 and Fig. 2). In the other two of
seven patients with CD4 as effectors, there was a slightly
different hierarchy of antigen recognition. Cytotoxicity
targeted at the VvgD1 recombinant was highest followed by
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DFH
Z. Mikloska and A. L. Cunningham
VvgC, then VvgB and VvgH (Fig. 2). In each patient the
differences in individual glycoprotein recognition in either
of the two hierarchies gD " gC " gB " gH or gD "
gB " gC " gH were significantly different from each other
(P ! 0±05, Student’s t-test). CD4 and CD4 and NK cell cytotoxicity for VvgH-infected keratinocytes was lowest in eight
of nine patients (for CD4 and NK cells mean net specific lysis
of 30 %, 26 % for CD4 T cells). In one of nine patients there
was no significant difference in cytotoxicity for gC and
gH. The reproducibility of CD4 lymphocyte cytotoxicity was
evaluated by repeating the experiments in the one patient
on five different occasions (with three autologous split skin
grafts taken at different times). The hierarchy and mean net
specific lysis for each glycoprotein were very similar.
CD4 T-cell cytotoxicity for HSV and vaccinia virus–HSV
glycoprotein recombinants was markedly inhibited by incubation with anti HLA-DR monoclonal antibody (as shown in
Fig. 3) indicating that this cytotoxicity was MHC class IIrestricted. Spontaneous release of &"Cr in all cytotoxicity and
the antibody-blocking experiments was always less than 15 %
of the total release from the keratinocyte cultures.
PBMC, CD4 and NK and CD4 T-cell cytotoxicity for
IFN-γ-pretreated keratinocytes infected with HSV-1
(positive) and vaccinia virus (negative) controls
PBMC were used as effectors in five different donors. In
these experiments, mean specific lysis for HSV-1-infected
keratinocytes was 72 % compared with mean specific lysis of
16 % for vaccinia virus-infected keratinocytes.
In all six patients in whom CD4 and NK cells were used as
effectors there was marked and significantly (P ! 0±05) higher
lysis for HSV-1-infected than for vaccinia virus controlinfected, IFN-γ-pretreated keratinocytes (Table 4). Mean net
lysis for HSV-1 was 76 % and for vaccinia virus controls 18 %.
In seven patients with CD4 cells as effectors the mean net lysis
for HSV-1-infected keratinocytes was 65 % and for the vaccinia
virus control 18 %. In all but one patient, CD4 and NK-cell
cytotoxicity for gH was significantly greater than the vaccinia
virus control.
CD4 T-lymphocyte cytotoxicity for VP16 mutants
CD4 T-lymphocyte cytotoxicity for IFN-γ-pretreated
keratinocytes infected with RP2 (HSV-2 VP16 exchange
mutant), RP3 or RP4 (VP16 partial deletion mutants) was
compared with that for the RP1 control under conditions of
equal m.o.i. and similar HSV glycoprotein expression (see
above). In two of nine patients a significant reduction in
cytotoxicity for the RP3 mutant compared with the parental
control was observed : 49 % and 56 % for the RP1 control and
32 % and 39 % for the RP3 VP16 mutant (results for one shown
in Fig. 4 a). In the other seven there was no significant
difference in cytotoxicity between the RP3 mutant and control
(results for one shown in Fig. 4 b). Another four patients were
DFI
(a)
(b)
Fig. 4. Comparison of CD4 cell cytotoxicity for IFN-γ-pretreated
keratinocytes infected with the partial deletion mutants RP3 or parental
HSV-KOS (RP1) control or mock-infected (cell control). Representative
experiments : (a) significant reduction in cytotoxicity of RP3 mutant virus
in comparison with RP1 control (a similar result was obtained in another
patient), and (b) no significant reduction in cytotoxicity of RP3 mutant
virus in comparison with RP1 control (similar results were obtained in 11
other patients). Effector to target ratio was 80 : 1. Cell control, mockinfected keratinocytes.
tested for specific cytotoxicity against RP2 and RP4 in
comparison with RP1. No reduction in specific cytotoxicity
was observed. This indicates that the epitope bound by the
RP3 (and larger RP4) deletion is occasionally important as a
CTL epitope in some patients.
Discussion
In this autologous, human in vitro system, HSV antigenrestimulated cytotoxic T cells, within mononuclear populations
depleted of CD8 T lymphocytes and}or NK cells, consistently
recognized and lysed IFN-γ-stimulated, vaccinia virus–HSV
glycoprotein recombinant-infected keratinocytes expressing
the HSV glycoproteins gB, gC and gD with lesser and variable
recognition of gH. These cells also recognized the abundant
HSV-1 tegument protein VP16 expressed in keratinocytes
stimulated with IFN-γ in a minority of patients as shown by
infection with the VP16 partial deletion mutant RP3. In all
three cell populations (mononuclear cells, CD4 and NK cells
and CD4 T cells) from HSV-seropositive patients there was
almost always a similar hierarchy of recognition of the proteins.
In the CD4 T-lymphocyte subset, cytotoxicity was usually
greatest for gD, followed by gB, gC with lesser and variable
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HSV-1 targets for human CD4 cytotoxicity
recognition of gH and an epitope in VP16 (at all effector to
target ratios). In two of seven patients cytotoxicity for gC was
greater than for gB.
A significant decrease in CD4 T-lymphocyte cytotoxicity
was detected in two of 13 patients when their keratinocytes
were infected with the RP3 or RP4 mutants with the partial
deletions of 7 % and 16 % of the VP16 protein (amino acids
456–490 for RP3 and 413–490 for RP4) instead of the parental
KOS strain. Therefore, recognition of this VP16 region as an
important epitope occurs in a minority of human subjects. In
future experiments, use of vaccinia virus VP16 recombinants
may demonstrate wider recognition of VP16 epitopes.
The similarity in hierarchy of recognition of glycoproteins
with all subsets consisting of CD4 lymphocytes and NK cells
or total PBMC, reflected both the predominance of CD4
lymphocyte cytotoxicity in this system, and also the predominant recognition of late HSV protein targets in IFN-γstimulated, HLA-DR antigen-expressing keratinocytes which
we recently reported (Mikloska et al., 1996). CD4 T lymphocytes recognized HSV late proteins predominantly and
consistently. CD8 T-lymphocyte cytotoxicity was only detectable with IFN-γ-stimulated keratinocytes and predominantly targeted early proteins. In these experiments, NK-cell
cytotoxicity was also predominantly directed at early proteins,
as previously reported by others (Fitzgerald-Bocarsly et al.,
1991). Macrophage cytotoxicity was negligible. We demonstrate here a predominance of CD4 T-lymphocyte cytotoxicity for major late structural proteins of the virion,
including the HSV glycoproteins gB, gC, gD and gH and
occasionally the major tegument protein VP16. These results
suggest that there is a variety of immunodominant epitopes
within each of these proteins recognized by different MHC
class II types thus resulting in recognition of each protein by a
large proportion of the population. These results will be
enhanced by more focused future studies of CD4 T-cell clones
for individual peptide epitopes in HLA-DR-typed individuals.
The difference between CD4 and CD8 lymphocyte specificity
for HSV glycoproteins (and IE}E proteins) is probably due to
differences in intracellular processing and subsequent binding
to HLA-DR of the two classes of proteins, as reported for the
glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (Oxenius et al., 1995). The slightly higher
levels of cytotoxicity by mononuclear than CD4 cells and one
case of greater recognition of gC than gD in the current
experiments may reflect the lower level and variable inconstant
recognition of late HSV proteins by CD8 cytotoxic T
lymphocytes, as previously reported (Mikloska et al., 1996).
The low recognition of gH could be due to several factors
including low levels of transport to the cell membrane and
hence to the MHC class II processing pathway, in the absence
of gL. This is being investigated.
A potential confounding factor in establishing the hierarchy
of glycoprotein recognition could be differences in the
proportions of keratinocytes infected with recombinant
vaccinia virus expressing the different HSV glycoproteins. To
ensure a lack of bias in target protein recognition, the vaccinia
virus–HSV glycoprotein recombinants were added at the same
input m.o.i. as each target epidermal cell monolayer, and the
proportion and intensity of cells staining for the target protein
were assessed by immunofluorescence at different times. Flow
cytometry using permeabilized keratinocytes with intact
antigen expression was found to be difficult, mainly because of
the obstacles in dissociating the cells tightly bound by
desmosomes. However, similar high proportions of keratinocytes stained for the glycoproteins gB, gC and gD at similar
high intensities of staining at the relevant time for cytotoxicity,
14–18 h post-infection. The proportion of cells expressing gH
was lower than for the other glycoproteins (despite the same
m.o.i.) and may partially, but not completely, explain the lower
levels of cytotoxicity for this glycoprotein. Therefore the
results presented in Table 1 do not account for the hierarchy in
glycoprotein recognition and, given the high proportion and
intensities of staining, these conclusions are unlikely to be
changed by the more accurate quantitative flow cytometric
techniques.
In the restimulation process, inactivated whole HSV antigen
was added to PBMC. Therefore any bias to recognition of
different proteins would be determined by the relative
proportions of the proteins themselves within the virusinfected cells and differences in their processing within antigenpresenting cells (APC) such as macrophages (reflecting the
same processes and biases in recurrent lesions in vivo).
Furthermore, although it is true that this type of restimulation
skews the response towards CD4 CTL as opposed to
restimulation with HSV-infected (IFN-γ-stimulated) keratinocytes, this is the likely scenario in the stages of early lesions in
vivo as shown by the following sequence. HSV infects
keratinocytes from axon termini, down-regulating MHC class
I before cell lysis and release of the full repertoire of viral
proteins. Uptake and presentation of HSV antigens by MHC
class II-expressing resident dendritic cells and resident}
infiltrating macrophages to infiltrating CD4 cells leads to
induction of cytotoxicity and IFN-γ production. Only after
restoration of MHC class I expression on keratinocytes by
IFN-γ can they act as stimulators to memory CD8 T cells.
As discussed in our previous papers, bulk cytotoxicity
assays combined with lymphocyte subset deletion have been
used in these initial studies because of the impracticality of
using cytotoxic T-cell precursor limiting dilution assays
(Cunningham & Noble, 1989 ; Mikloska et al., 1996). The
number of cultured keratinocytes which can be obtained even
from split skin grafts in patients undergoing minor operations
is insufficient to allow appropriate numbers of replicates for
these limiting dilution assays.
Furthermore, the use of IFN-γ-stimulated, HSV-infected
keratinocytes as stimulators as well as targets is technically
very difficult and probably relevant only to the later stages of
the lesion. Also, T-cell cloning is selective and hence we would
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Z. Mikloska and A. L. Cunningham
not obtain sufficient breadth in our sampling of the human
outbred population to determine whether the results on HSV
protein target recognition are representative.
Despite the marked down-regulation of MHC class I
antigen expression on cells by HSV, partial reconstitution by
IFN-γ (which also up-regulates TAP expression in infected
cells) (Fruh et al., 1995 ; Hill et al., 1995) is a host cell counter to
this mechanism of immune evasion (Mikloska et al., 1996).
Hence CD8 T-lymphocyte cytotoxicity may still play an
important role in recurrent herpes simplex lesions of at least
some patients (Posavad et al., 1996) although this may vary in
importance as, on average, HSV titres in lesions have already
declined when CD8 T-cell influx is maximal, after 2–3 days
(Spruance et al., 1977 ; Cunningham et al., 1985). Nevertheless,
further attention should be paid to the herpes simplex protein
target recognition by CD8 T lymphocytes in IFN-γ-stimulated
keratinocytes and this is currently under investigation. There is
a large body of work on protein target recognition by CD8 T
lymphocytes and T-lymphocyte clones in mice (Lawman et al.,
1980 ; Martin et al., 1988 ; Schmid & Rouse, 1992). It will be
interesting to compare the relative target recognition of
human CD8 cytotoxic T lymphocytes in IFN-γ-stimulated
targets with these results in mice. HSV does not appear to
down-regulate MHC class I in mice because HSV ICP47 fails to
complex with murine cell TAP (York et al., 1994 ; Fruh et al.,
1995).
Nash et al. (1987) also demonstrated the importance of CD4
lymphocytes in primary HSV infection of mice using monoclonal antibody depletion.
The in vitro model used in these experiments contains the
most appropriate target cell for cytotoxicity, the HSV-1infected keratinocyte, which is stimulated by IFN-γ to express
HLA-DR (Basham et al., 1984, 1985) and enhanced MHC class
I antigens. This reflects what actually occurs in the lesion in
vivo (Cunningham et al., 1985). It would be ideal, but extremely
difficult, to use the CD4 lymphocytes infiltrating the lesion as
the effector cells and also HSV-infected autologous HLA-DR
antigen-expressing epidermal cells as the targets in these
assays. Koelle et al. (1994 a, b) cloned HSV-2-specific CD4 T
lymphocytes from the lesions of recurrent herpes (with PHA)
but used Epstein–Barr virus-transformed lymphoblastoid cells
as APC or targets. Somewhat surprisingly they found evidence
of limited T-cell reactivity to the glycoproteins gB, gC and gD,
which differed from our results and those of Zarling et al.
(1986), probably partly due to lack of recognition of common
epitopes between gC1 and gC2 and the nature and concentrations of recombinant antigens used. However, the most
important differences were probably in target cells and also in
HSV antigen-processing in lesions compared with that in
blood cells.
The results of this study have considerable implications for
the development of herpes simplex vaccines. The concept of
therapeutic vaccines has been established experimentally in
guinea-pig models. Stanberry (1992) has demonstrated that the
DGA
immunization of guinea-pigs with recombinant gB, gD or both
gB and gD after the experimental establishment of recurrent
genital herpes reduces the frequency of genital herpes lesions.
Although Straus et al. (1994) also reported a significant overall
reduction in lesions of 20 % after immunization of humans with
a recombinant gB}gD vaccine, whether this can be repeated
with recently developed adjuvants which increase the stimulation of T-lymphocyte activity and}or cytotoxic T-cell
activity [(MF59), monophosphoryl lipid A (MPL)] awaits the
results of current clinical trials. However, the addition of MPL
to an HSV gD vaccine candidate has clearly shown a marked
increase in CD4 lymphocyte reactivity to the candidate
immunogen (Bastin et al., 1994).
If there is indeed a difference in HSV antigen specificity
between restimulated blood and lesional CD4 cytotoxic
lymphocytes directed at infected epidermal cells and if these
blood lymphocytes home to recurrent herpetic lesions this
may explain the variability in the response to therapeutic
immunization and help in prediction of responses to future
prophylactic vaccines.
We thank Professor B. Rouse and Professor A. Minson for vaccinia
virus–HSV glycoprotein recombinants and monoclonal antibodies, Dr S.
Triezenberg for RP1 virus and RP2–RP4 mutants. The research was
supported by a grant from the National Health and Medical Research
Council of Australia.
References
Basham, T. Y., Nickoloff, B. J., Merigan, T. C. & Morhenn, V. B. (1984).
Recombinant γ interferon induces HLA-DR expression on cultured
human keratinocytes. Journal of Investigative Dermatology 83, 88–90.
Basham, T. Y., Nickoloff, B. J., Merigan, T. C. & Morhenn, V. B. (1985).
Recombinant gamma interferon differentially regulates class II antigen
expression and biosynthesis on cultured human keratinocytes. Journal of
Interferon Research 5, 23–32.
Bastin, C., Hermand, P., Francotte, M., Garcon, N., Slaoui, M. & Pala,
P. (1994). Synergistic association of adjuvants QS21 and MPL for
induction of cytolytic T lymphocytes and T helper responses to
recombinant protein antigens. In Abstracts of the 34th Interscience Conference
on Antimicrobial Agents and Chemotherapy of the American Society for
Microbiology. Abstr. G41. Washington, DC : American Society for
Microbiology.
Cantin, E. M., Eberle, R., Boldick, J. L., Moss, B., Willey, D. E., Notkins,
A. R. & Openshaw, H. (1987). Expression of HSV glycoprotein B by a
recombinant vaccinia virus and protection of mice against lethal HSV-1
infection. Proceedings of National Academy of Sciences, USA 84, 5908–5912.
Cress, W. D. & Triezenberg, S. J. (1990). Critical structural elements of
the VP16 transcriptional activation domain. Science 251, 87–90.
Cunningham, A. L. & Merigan, T. C. (1983). Gamma interferon
production appears to predict time of recurrence of herpes labialis. Journal
of Immunology 130, 2397–2400.
Cunningham, A. L. & Merigan, T. C. (1984). Leu3 T-cells produce
gamma-interferon in patients with recurrent herpes labialis. Journal of
Immunology 132, 197–202.
Cunningham, A. L. & Noble, J. R. (1989). Role of keratinocytes in
human recurrent herpetic lesions. Journal of Clinical Investigation 83,
490–496.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 18:01:56
HSV-1 targets for human CD4 cytotoxicity
Cunningham, A. L., Turner, R. R., Miller, A. C., Para, M. F. & Merigan,
T. C. (1985). Evolution of recurrent herpes simplex lesions : an
immunohistologic study. Journal of Clinical Investigation 75, 226–233.
Fauci, A. S. (1988). The human immunodeficiency virus : infectivity and
mechanisms of pathogenesis. Science 239, 617–622.
Fitzgerald-Bocarsly, P., Howell, D. M., Pettera, L., Tehrani, S. &
Lopez, C. (1991). Immediate-early gene expression is sufficient for
induction of natural killer cell-mediated lysis of herpes simplex virus type
1 infected fibroblasts. Journal of Virology 65, 3151–3160.
Forrester, A. J., Sullivan, V., Simmons, A., Blacklaws, B. A., Smith, G.
L., Nash, A. A. & Minson, A. C. (1991). Induction of protective immunity
with antibody to herpes simplex virus type 1 glycoprotein H (gH) and
analysis of the immune response to gH expressed in recombinant vaccinia
virus. Journal of General Virology 72, 369–375.
Fruh, K., Ahn, K., Djaballah, H., Sempe, P., van Endert, P. M., Tampe,
R., Peterson, P. A. & Yang, Y. (1995). A viral inhibitor of peptide
dogenous viral proteins in association with major histocompatibility
complex II : on the role of intracellular compartmentalization, invariant
chain and the TAP transporter system. European Journal of Immunology
25, 3402–3411.
Posavad, C. M. & Rosenthal, K. L. (1992). Herpes simplex virusinfected human fibroblasts are resistant to and inhibit cytotoxic Tlymphocyte activity. Journal of Virology 66, 6264–6272.
Posavad, C. M., Newton, J. J. & Rosenthal, K. L. (1993). Inhibition of
human CTL-mediated lysis by fibroblasts infected with herpes simplex
virus. Journal of Immunology 151, 4865–4873.
Posavad, C. M., Koelle, D. M. & Corey, L. (1996). High frequency of
CD8 cytotoxic T lymphocyte precursors specific for herpes simplex
viruses in persons with genital herpes. Journal of Virology 70, 8165–8168.
Reeves, W. C., Corey, L., Adams, H. G., Vontver, L. A. & Holmes, K. K.
(1981). Risk of recurrence after first episodes of genital herpes. New
England Journal of Medicine 305, 315–319.
transporters for antigen presentation. Nature 375, 415–418.
Schmid, D. S. & Rouse, B. T. (1992). The role of T cell immunity in
Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh,
H. & Johnson, D. C. (1995). Herpes simplex virus turns off TAP to
control of herpes simplex virus. In Herpes Simplex Virus. Pathogenesis,
Immunobiology and Control, pp. 57–74. Edited by B. T. Rouse. Berlin :
Springer-Verlag.
evade host immunity. Nature 375, 411–415.
Koelle, D. M., Tigges, M. A., Burke, R. L., Symington, F. W., Ridell,
S. R., Abbo, H. & Corey, L. (1993). Herpes simplex virus infection of
human fibroblasts and keratinocytes inhibits recognition by cloned CD8
cytotoxic T-lymphocytes. Journal of Clinical Investigation 91, 961–968.
Koelle, D. M., Abbo, H., Peck, A., Ziegweid, K. & Corey, L. (1994 a).
Direct recovery of herpes simplex virus (HSV)-specific T-lymphocyte
clones from recurrent genital HSV-2 lesions. Journal of Infectious Diseases
169, 956–961.
Koelle, D. M., Corey, L., Burke, R. L., Eisenberg, R. J., Cohen, G. H.,
Pichyangkura, R. & Triezenberg, S. J. (1994 b). Antigenic specificities
of human CD4 T cell clones recovered from recurrent genital herpes
simplex virus type 2 lesions. Journal of Virology 68, 2803–2810.
Lawman, J. R., Rouse, B. T., Courtney, R. J. & Walker, R. D. (1980).
Siegal, F. P., Lopez, C., Hammer, G. S., Brown, A. E., Kornfeld, S. J.,
Gold, J., Hassett, J., Hirschman, S. Z., Cunningham-Rundles, C. &
Adelsberg, B. R. (1981). Severe acquired immunodefiecency in male
homosexuals, manifested by chronic perianal ulcerative herpes simplex
lesions. New England Journal of Medicine 305, 1439–1444.
Spruance, S. L., Overall, J. C., Kern, E. R., Krueger, G. G., Pliam, V. &
Miller, W. (1977). The natural history of recurrent herpes simplex
labialis. New England Journal of Medicine 297, 69–75.
Stanberry, L. R. (1992). Pathogenesis of herpes simplex virus infection
and animal models for its study. In Herpes Simplex Virus. Pathogenesis,
Immunobiology and Control, pp. 15–30. Edited by B. T. Rouse. Berlin :
Springer-Verlag.
Straus, S. E., Corey, L., Burke, R. L., Savarese, B., Barnum, G., Krause,
P. R., Kost, R. G., Meier, J. L., Sekulovich, R., Adair, S. F. and others
(1994). Placebo-controlled trial of vaccination with recombinant
Cell-mediated immunity against herpes simplex virus : induction of
cytotoxic T lymphocytes. Infection and Immunity 27, 133–139.
Martin, S., Courtney, R. J., Fowler, G. & Rouse, B. T. (1988). Herpes
simplex virus type 1 specific cytotoxic T lymphocytes recognize viral
nonstructural proteins. Journal of Virology 62, 2265–2273.
Martin, S., Mercadal, C. M., Weir, J. P. & Rouse, B. T. (1993). The
proportion of herpes simplex virus specific cytotoxic T lymphocytes (Tc)
that recognise glycoprotein C varies between individual mice and is
dependent on the form of immunisation. Viral Immunology 6, 21–33.
glycoprotein D of herpes simplex virus type 2 for immunotherapy of
genital herpes. Lancet 343, 1460–1463.
Torseth, J. W. & Merigan, T. C. (1986). Significance of local gamma
interferon in recurrent herpes simplex infection. Journal of Infectious
Diseases 153, 979–984.
Ward, P. L. & Roizman, B. (1994). Herpes simplex genes : the blueprint
of a successful human pathogen. Trends in Genetics 10, 267–274.
Mikloska, Z., Kesson, A. M., Penfold, M. E. T. & Cunningham, A. L.
(1996). Herpes simplex virus protein targets for CD4 and CD8
Weinheimer, S. P., Boyd, B. A., Durham, S. K., Resnick, J. L. & O’Boyle,
D. R. (1992). Deletion of VP16 open reading frame of herpes simplex
lymphocyte cytotoxicity in cultured epidermal keratinocytes treated
with interferon-γ. Journal of Infectious Diseases 173, 7–17.
Nash, A. A., Jayasuriya, A., Phelan, J., Cobbold, S. P., Waldmann, H. &
Prospero, T. (1987). Different roles for L3T4+ and Lyt 2+ T cell subsets
in the control of an acute herpes simplex virus infection of the skin and
nervous system. Journal of General Virology 68, 825–833.
Overall, J. C., Spruance, S. L. & Green, J. A. (1981). Viral-induced
leukocyte interferon in vesicle fluid from lesions of recurrent herpes
labialis. Journal of Infectious Diseases 143, 543–547.
Oxenius, A., Bachmann, M. F., Ashton-Rickardt, P. G., Tonegawa, S.,
Zinkernagel, R. M. & Hengartner, H. (1995). Presentation of en-
virus type 1. Journal of Virology 66, 258–269.
York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L. &
Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits
antigen presentation to CD8 T-lymphocytes. Cell 77, 525–535.
Zarling, J. M., Moran, P. A., Burke, R. L., Pachl, C., Berman, P. W. &
Lasky, L. A. (1986). Human cytotoxic T-cells directed against herpes
simplex virus-infected cells. IV. Recognition and activation by cloned
glycoproteins gB and gD. Journal of Immunology 136, 4669–4673.
Received 30 June 1997 ; Accepted 23 September 1997
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