Download Reduced immune responses after vaccination with a recombinant

Journal of General Virology (2005), 86, 2401–2410
DOI 10.1099/vir.0.81104-0
Reduced immune responses after vaccination with
a recombinant herpes simplex virus type 1 vector in
the presence of antiviral immunity
Henning Lauterbach,13 Christine Ried,1 Alberto L. Epstein,2
Peggy Marconi3 and Thomas Brocker1
1
Institute for Immunology, Ludwig Maximilians University Munich, Goethestrasse 31, 80336
Munich, Germany
Correspondence
Thomas Brocker
[email protected]
2
University Claude-Bernard Lyon 1, Centre de Genetique Moleculaire et Cellulaire, Lyon,
France
3
University of Ferrara, Department of Experimental and Diagnostic Medicine, Ferrara, Italy
Received 13 April 2005
Accepted 14 June 2005
Due to the continuous need for new vaccines, viral vaccine vectors have become increasingly
attractive. In particular, herpes simplex virus type 1 (HSV-1)-based vectors offer many
advantages, such as broad cellular tropism, large DNA-packaging capacity and the induction of
pro-inflammatory responses. However, despite promising results obtained with HSV-1-derived
vectors, the question of whether pre-existing virus-specific host immunity affects vaccine efficacy
remains controversial. For this reason, the influence of pre-existing HSV-1-specific immunity on
the immune response induced with a replication-defective, recombinant HSV-1 vaccine was
investigated in vivo. It was shown that humoral as well as cellular immune responses against a
model antigen encoded by the vaccine were strongly diminished in HSV-1-seropositive mice. This
inhibition could be observed in mice infected with wild-type HSV-1 or with a replication-defective
vector. Although these data clearly indicate that pre-existing antiviral host immunity impairs the
efficacy of HSV-1-derived vaccine vectors, they also show that vaccination under these constraints
might still be feasible.
INTRODUCTION
Herpes simplex virus type 1 (HSV-1) is the causative agent
of a variety of diseases [reviewed by Stanberry et al. (2000)].
However, in recent decades it has become possible to
exploit the advantageous features of HSV-1 for gene
delivery, by stepwise elimination of viral pathogenicity
[reviewed by Advani et al. (2002)]. HSV-1-derived vectors,
including replication-deficient and infectious single-cycle
(DISC) vectors or amplicons, offer many advantages over
other viral vectors for gene-therapeutic and vaccine approaches [reviewed by Thomas et al. (2003)]. They can transduce
a wide range of cell types, including non-dividing
cells (Coffin et al., 1998; Mikloska et al., 2001), show high
transduction efficiency (Moriuchi et al., 2000) and have
large DNA-packaging capacities (Krisky et al., 1998; WadeMartins et al., 2003). Further, their genomes persist in the
cell nucleus as extrachromosomal episomes (Mellerick &
Fraser, 1987) and therefore should not induce mutagenesis
by DNA insertion in transduced cells (Li et al., 2002). The
many applications for HSV-1-derived vectors have recently
been reviewed elsewhere (Burton et al., 2001).
3Present address: The Scripps Research Institute, Division of Virology,
Department of Neuropharmacology, La Jolla, CA, USA.
0008-1104 G 2005 SGM
Upon HSV-1 infection, innate immunity provides the first
line of defence, with activated macrophages, dendritic
cells (DC), NK cells and cd T cells as key players (Bukowski
et al., 1994; Kadowaki et al., 2000; Kodukula et al., 1999;
Siegal et al., 1999). Together with cytokines and the complement cascade, these cells limit the spread of epidermal
infection by the herpesvirus (Ahmad et al., 2000; Da Costa
et al., 1999; Feduchi et al., 1989; Melchjorsen et al., 2002).
Neutralizing antibodies specific for the major envelope
glycoproteins gB, gD and gH/L, as well as CD4+ and CD8+
T lymphocytes recognizing HSV antigens (Ags) can be
detected after HSV-1 infection (Mikloska & Cunningham,
1998; Mikloska et al., 1996). Despite this HSV-specific
immunity, wild-type (wt) HSV can persist life-long in the
host due to different viral mechanisms that interfere with
immune recognition (Fries et al., 1986; Hill et al., 1995;
Johnson & Feenstra, 1987; Samady et al., 2003; Sloan et al.,
2003) and due to its ability to establish latent infections in
sensory neurons.
In animal studies, the use of replication-defective HSV
vaccines has been shown to induce robust and long-lived
HSV-specific immunity (Morrison & Knipe, 1996, 1997).
In addition, many recombinant HSV vectors expressing
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H. Lauterbach and others
foreign Ags have been developed for gene-therapeutic
approaches (Huard et al., 1995; Palmer et al., 2000) and
vaccination against bacterial (Lauterbach et al., 2004) or
viral (Hocknell et al., 2002; Murphy et al., 2000) infection. A
major concern affecting the potential use of recombinant
HSV vectors is the possible impairment of vaccine efficacy
by pre-existing anti-HSV immunity. While this has been
reported for adenovirus and poxvirus vectors (Etlinger
& Altenburger, 1991; Papp et al., 1999; Parr et al., 1998;
Schulick et al., 1997), the vaccine efficiency of poliovirus(Mandl et al., 2001) as well as alphavirus (Pushko et al.,
1997)-based vectors seems not to be impaired in immune
hosts.
In light of the high (60–90 %) prevalence of HSV-1 infection
among the adult population (Cunningham et al., 2000;
Stanberry et al., 2000), it is particularly important to
investigate the effect of pre-existing immunity on HSVvaccine efficiency. It has recently been shown that upon
immunization with a replication-defective HSV-1 vector,
the Ag-specific antibody (Ab) response is long-lasting and
not impaired by prior HSV exposure (Brockman & Knipe,
2002). Similar results have been obtained in HSV-mediated
oncolytic therapy (Chahlavi et al., 1999). However,
gene transfer into experimental brain tumours in HSV-1seropostive mice was less efficient (Herrlinger et al., 1998).
Likewise, an HSV-1 amplicon vaccine induced immune
responses that were reduced by prior infection with HSV-1
(Hocknell et al., 2002).
Since these studies have given contradictory results concerning the inhibitory effects of pre-existing HSV-1
immunity (Brockman & Knipe, 2002; Chahlavi et al.,
1999; Herrlinger et al., 1998; Hocknell et al., 2002), we
investigated the Ag-specific humoral and cellular immune
response induced by replication-defective, recombinant
HSV-1 in seropositive mice. We report that pre-existing
HSV-1 immunity does substantially reduce the ability of an
HSV-1-derived vaccine to induce Ag-specific humoral and
cellular immune responses. Furthermore, we show that the
negative impact is similar in mice infected either with
homologous or heterologous HSV-1 wt strains or with a
homologous replication-defective virus.
METHODS
Mice. All mice were bred and maintained under standard condi-
tions in the animal facilities of the Institute for Immunology,
Ludwig Maximilians University Munich, Germany, or the Department of Pharmacy, University of Ferrara, Italy. OT-1 mice [expressing transgenic T-cell receptor (TCR) specific for chicken ovalbumin
(OVA)257-263/MHC class I Kb] (Hogquist et al., 1994) and rat insulin
promoter (RIP)-OVAlo mice expressing OVA in b-islet cells of the
pancreas (Kurts et al., 1999) have been described previously.
Viruses. In order to infect mice with non-lethal doses of wt HSV-1,
26106 p.f.u. HSV-1 KOS or strain F were injected intravenously (16107 p.f.u. ml21 in PBS). Preparation of OVA-encoding,
replication-deficient HSV-1 vaccine vectors (T0H-OVA) and green
fluorescent protein (GFP)-encoding control vectors (T0-GFP) has
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been described previously (Lauterbach et al., 2004). These vectors
harbour deletions in three immediate-early genes (ICP4 and ICP27,
which are essential for virus replication, and ICP22). lacZ was
inserted into the UL41 locus, which led to a disruption of this gene,
which encodes the virus host shut-off (vhs) protein. For recombinant HSV-1 (rHSV-1) vaccination, frozen virus stocks were thawed
on ice, and diluted in PBS to 46106 rHSV-1 (200 ml)21 for intravenous (i.v.), intraperitoneal (i.p.) and subcutanous (s.c.) injection,
and to 46106 rHSV-1 (50 ml)21 for intradermal (i.d.) injection.
Before injection, virus suspensions were sonicated in a water bath
for 5 s.
Adoptive transfer. OT-1 CD8+ T cells were prepared from lymph
nodes and spleens of transgenic mice. Briefly, spleen and lymph
nodes were taken out, and single-cell suspensions were prepared.
Erythrocytes were removed by osmotic lysis, and after determining
the percentage of TCR-transgenic T cells by flow cytometry, 16106
transgenic T cells were injected intravenously into recipient mice.
Enzyme-linked immunosorbent assay (ELISA). For the detec-
tion of OVA-specific antibodies, 96-well microtitre plates (Nunc
Maxisorp) were coated with 15 mg chicken OVA ml21 (Sigma
Chemicals Co.) at room temperature overnight. For the detection of
anti-HSV antibodies, plates were either coated with T0-GFP in PBS
(26105 p.f.u. ml21) overnight at 4 uC or with HSV-1 MacIntyre
viral lysate (tebu-bio GmbH) under the same conditions. Plates
were blocked (PBS, 0?5 % milk powder and 0?05 % NaN3), and
immune sera (diluted 1 : 100 in blocking buffer) were incubated for
2 h at room temperature. After washing five times with PBS, HRPlabelled second-step goat sera specific for mouse IgG (Serotec) in
PBS, 0?5 % milk powder, 0?05 % Tween 20, were added and incubated for 2 h. After five washing steps, the amount of bound Ab
was determined by the addition of substrate solution (1 mM
3,39,5,59 tetramethylbenzidine, 0?3 ml ml21 30 % H2O2, in 0?2 M
potassium acetate). The reaction was stopped by the addition of 2 N
H2SO4, and the A450 was determined with a Vmax-microplate reader
(Molecular Devices Corporation).
Monoclonal
antibodies,
tetramers
and
flow
cytometry.
Lymphocytes were analysed using the monoclonal antibody (mAb)
anti-CD8a-APC (Ly2) from Caltag (Burlingame, CA). Analytic flow
cytometry was performed on a FACScalibur (Becton Dickinson),
and the data were analysed using Cellquest software (Becton
Dickinson). H-2Kb/OVA257-264-phycoerythrin (PE) and H-2Kb/
gB498-505-tetramer-PE complexes were purchased from ProImmune
Limited.
In vivo cytotoxic T-lymphocyte (CTL) assay. This assay was per-
formed as published previously (Kleindienst et al., 2005). Syngeneic
C57BL/6 spleen and lymph-node cells were depleted of erythrocytes
by osmotic lysis. Cells were washed and split into two populations.
One population was pulsed with 1026 M OVA257-264-peptide for 1 h
at 37 uC, washed and labelled with a high concentration of carboxyfluorescein diacetate succinimidyl ester (CFSE, 2?5 mM) (CFSEhigh
cells). The second control population was labelled with a low concentration of CFSE (0?25 mM) (CFSElow cells). For i.v. injection, an
equal number of cells from each population (CFSEhigh and CFSElow)
was mixed, such that each mouse received a total of 26107 cells.
Cells were injected into vaccinated mice. Twenty hours later, mice
were sacrificed and spleen and lymph nodes removed. Cell suspensions were analysed by flow cytometry; approximately 56105
CFSE-positive cells were collected for analysis. Peptide-pulsed and
non-pulsed target cells were recognized according to their different
CFSE intensities. To calculate specific lysis, the following formulae
were used: ratio=(percentage CFSElow/percentage CFSEhigh); percentage specific lysis=[12(ratio unprimed/ratio primed)6100].
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Journal of General Virology 86
HSV-based vaccines in HSV-seropositive hosts
RESULTS
A recombinant, replication-defective HSV-1 vector encoding OVA, T0H-OVA, was chosen as a model vaccine in these
studies. This vaccine was previously shown to induce strong
OVA-specific CTL responses and protected mice against
infection with lethal doses of intracellular bacteria (Listeria
monocytogenes) (Lauterbach et al., 2004). For clarity, the
term vaccination/immunization will be used throughout
the manuscript with respect to the inoculation of mice
with T0H-OVA to induce specific immune responses. The
homologous vector not encoding the Ag OVA, T0-GFP,
and the HSV-1 wt strains KOS and F, were employed to
induce seroconversion in mice. Both replication-defective
vectors T0H-OVA and T0-GFP are negative for ICP4,
ICP22, ICP27 and vhs, and are based on a HSV-1 KOS
backbone (Lauterbach et al., 2004).
OVA-specific humoral responses are reduced or
inhibited completely in HSV-seropositive mice
It has been shown elsewhere that induction of long-lasting
specific Ab responses after immunization with HSV vectors
is possible, even in the presence of anti-HSV antibodies
(Brockman & Knipe, 2002). To test the ability of T0H-OVA
to induce an OVA-specific Ab response in HSV-seropositive
animals, we infected mice with T0-GFP by the i.v. or s.c.
route (Fig. 1a and b), since replication-defective HSV-1
mutants have already been shown to induce antiviral
immunity (Farrell et al., 1994; Geiss et al., 2000; Morrison &
Knipe, 1994). The injection routes were chosen according to
results obtained by infecting mice via different routes (data
not shown). i.v. and s.c inoculations were performed in
order to induce high and low anti-HSV-1 Ig titres, respectively. The seroconversion was verified by measuring
anti-HSV Ab titres in the serum of infected mice 2 and
4 weeks after T0-GFP infection. In two independent experiments, we could confirm our previous observations that low
levels of anti-HSV-1 IgG were obtained via the s.c. route,
while i.v.-inoculated animals produced high levels of HSVspecific Ab (Fig. 1b, e). Four weeks post-infection with T0GFP, we vaccinated mice with T0H-OVA by the s.c. (Fig. 1a,
b, c) or i.v. (Fig. 1d, e, f) route. In both experiments OVAspecific IgG titres could be detected at 2 weeks after T0HOVA immunization in naive control animals (Fig. 1c, f,
6 weeks post-infection). However, in HSV-seropositive
mice, the detectable OVA-specific Ab titres were inversely
proportional to pre-existing HSV-specific Ab titres: mice
immunized by the s.c. route with T0H-OVA (Fig. 1a, b, c)
could not mount an OVA-specific humoral response, even if
pre-existing immunity created high (via i.v. inoculation) or
lower (via s.c. inoculation) titres of HSV-specific Ab. In
contrast, seropositive mice vaccinated by the i.v. route with
Fig. 1. Inhibition of Ab induction by pre-existing immunity. C57BL/6 mice were infected at day 0 with 46106 T0-GFP via the
indicated routes (a, d). Four weeks later, mice were immunized with 46106 T0H-OVA by either the s.c. (a) or i.v. (d) routes.
Sera were obtained every 2 weeks, diluted 1 : 50 and analysed by ELISA for anti-HSV IgG (b, e) (ELISA-plates were coated
with T0-GFP; see Methods) and anti-OVA IgG (c, f). Pre-immune status (day 0) from each group was determined with a pool
of sera. Results are expressed as the mean A450±SD from five mice per group.
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T0H-OVA still mounted a weak anti-OVA IgG response (Fig. 1f). The reduction was stronger in mice
that received i.v. T0-GFP and had higher anti-HSV serum
titres than animals with lower anti-HSV titres (Fig. 1e, f).
These results indicate that humoral responses to an HSV1-vector-encoded Ag are affected in HSV-1-seropositive
animals.
CTL expansion upon vaccination with rHSV-1 is
diminished by pre-existing immunity of the host
Previously, we had shown that T0H-OVA induces very
strong CD8+ T-cell responses (Lauterbach et al., 2004).
Here, we wanted to investigate the influence of pre-existing
anti-HSV-1 immunity on T0H-OVA-induced CTL responses. Mice were infected by the i.v. route with the control
vector T0-GFP, or left untreated. Three weeks after infection, seroconversion was verified by ELISA (data not
shown) and all mice were vaccinated with T0H-OVA.
OVA-specific CTL expansion was monitored by tetrameric
H-2Kb/OVA257-264 complexes (Tet), as shown in Fig. 2(a).
In non-immunized C57BL/6 mice, the frequency of
CD8+Tet+ T cells was between 0?02 and 0?05 % among
peripheral blood lymphocytes (PBLs) (Fig. 2a, left panel),
while in vaccinated mice an Ag-specific increase of
Tet+CD8+ T cells was detected (Fig. 2a, right panel). In
naive mice, the frequency of specific CD8+ T cells among
CD8+ PBLs increased 50-fold (from 0?11 % on day 0
to 5?53 % on day 13) after vaccination with T0H-OVA
(Fig. 2b). In contrast, mice that were already immune to
HSV-1 showed an extremely diminished OVA-specific
CD8+Tet+ T-cell expansion (5- to 10-fold increase,
Fig. 2b). In this group, we detected at day 13 after T0HOVA challenge only 0?63±0?38 % CD8+Tet+ T cells
among CD8+ PBLs (Fig. 2b). These data show that preexisting immunity in HSV-seropositive mice has a negative
influence on CD8+ T-cell responses.
Inhibition of CTL effector functions in the RIPOVAlo mouse model
In order to directly monitor the impact of pre-existing
HSV-1 immunity on the efficacy of an rHSV-1 vaccine, we
employed the well-described transgenic RIP-OVAlo mouse
model, in which OVA is expressed under the control of the
rat-insulin promoter in b-islet cells of the pancreas (Kurts
et al., 1999). In these mice, the CD8+ T-cell compartment
is ignorant of OVA, probably due to the insufficient
amount of cross-presentation (Kurts et al., 1999). Therefore,
TCR-transgenic, OVA-specific CD8+ T cells (OT-1 cells)
have been adoptively transferred into these mice. Immunization with a vaccine encoding OVA activates these Agspecific OT-1 cells, which, upon efficient activation, lyse
OVA-expressing pancreas cells. Consequently, these mice
develop diabetes-like symptoms, which can be directly
measured by an increase of glucose concentration in the
urine (Kurts et al., 1998; Nopora & Brocker, 2002). RIPOVAlo mice that had received adoptively transferred
OVA-specific CD8+ T cells (OT-1>RIP-OVAlo) were left
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Fig. 2. Inhibition of Ag-specific CTL expansion by pre-existing
immunity. C57BL/6 mice were infected with 46106 T0-GFP
by the i.v. route or not treated 21 days before i.p. vaccination
with 46106 T0H-OVA. Peripheral blood cells were stained
with anti-CD8-APC and H-2Kb/OVA257–264tetramers-PE and
analysed by flow cytometry. Representative dot plots of a naive
(a, left panel), a T0-GFP infected/T0H-OVA immunized (a,
middle panel) and a non-infected/T0H-OVA immunized (a, right
panel) mouse 13 days after vaccination are shown. The mean
percentage±SD of gated CD8+Tet+ CTL among PBLs is
indicated on each plot. (b) The frequencies of H-2Kb/
OVA257–264tetramer-specific CD8+ T cells in peripheral blood
were determined [as shown in (a)] by flow cytometry at the
days indicated and are displayed as the percentage of Tet+
cells of CD8+ PBL. Data represent mean±SD obtained from
four to five animals per group at each time point.
untreated or infected with T0-GFP via different routes.
Seventeen days later, we examined sera for the presence of
anti-HSV-1 antibodies. All mice infected by the i.v., i.p.
and i.d. routes were seropositive for anti-HSV IgG antibodies, whereas the mean IgG titre in s.c.-infected mice
was not significantly higher than in naive mice (Student’s t
test: P>0?5) (data not shown).
Nineteen days after T0-GFP infection, all OT-1>RIPOVAlo mice were immunized by the i.v. route with T0HOVA and monitored for the development of diabetes
as a direct read-out for in vivo induction of efficient
CTL responses (Fig. 3a). In the non-infected, seronegative group, all mice became diabetic after 5–6 days. Also, in
the group of mice that was s.c.-inoculated and showed
relatively modest anti-HSV Ab titres, 66 % of the mice
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HSV-based vaccines in HSV-seropositive hosts
Wt virus infection interferes with vaccine
efficiency
Fig. 3. Inhibition of CTL effector functions in the transgenic
RIP-OVAlo mouse model. RIP-OVAlo mice that had received
16106 adoptively transferred OT-1 CD8+ T cells were
infected with 46106 T0-GFP via the routes indicated or left
untreated. All mice were vaccinated 19 days later with 46106
T0H-OVA by the i.v. route. (a) Mice were monitored for induction of diabetes by measuring glucosuria, and considered diabetic when glucose concentration was ¢5?5 mmol l”1. (b) The
vaccine efficacy (percentage of diabetic mice at day 9 after
T0H-OVA vaccination) is shown in relation to the standardized
anti-HSV IgG titres (the mean IgG titre of seronegative mice
was defined as 1) (n=3 for each group).
became diabetic. In mice that had received i.d. or i.p. T0GFP, only one mouse per group could be considered as
diabetic at various days (open triangles and filled squares,
respectively). In contrast, vaccination with T0H-OVA did
not induce diabetes in previously i.v.-infected mice. In order
to see whether the amount of anti-HSV IgG correlated with
the grade of inhibition of the vaccine, we analysed IgG titres
in relation to the number of diabetic mice (Fig. 3b shows
as an example the data from day 9 after T0H-OVA immunization. Data from other time points show similar results).
This graph (Fig. 3b) demonstrates that the efficacy of T0HOVA immunization is negatively correlated to the magnitude of anti-HSV Ab titres, in other words, pre-existing
anti-HSV immunity diminishes the efficacy of a second
immunization with a homologous recombinant virus.
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In order to avoid recognition and subsequent elimination
by the host immune system, many immune evasion
mechanisms have evolved in HSV-1 (Friedman et al.,
2000; Nagashunmugam et al., 1998; Neumann et al., 2003;
Pollara et al., 2003). To test whether a ‘natural’ infection
with wt virus has similar inhibiting effects on a subsequent
vaccination with a recombinant vector (T0H-OVA), we
infected C57BL/6 mice with non-lethal doses of either
the wt viruses HSV-1 KOS and HSV-1 strain F or the
recombinant, replication-defective virus T0-GFP (see
Fig. 4a for experimental set-up and timing). We monitored
anti-HSV-1 IgG titre by ELISA using HSV-1 Ag from the
MacIntyre strain. At 14 and 26 days after infection, all
HSV-1-treated mice were positive for anti-HSV-1 IgG
(Fig. 4b). The titres in wt-infected mice were slightly higher
than in T0-GFP-infected mice. Furthermore, titres in these
mice increased over time, whereas the titres remained constant in animals infected with the non-replicating virus.
At days 6 and 29 after infection we monitored the frequency of CD8+ T cells specific for the immunodominant
HSV-1 glycoprotein (g)B498-505 peptide (Wallace et al.,
1999) using H-2Kb/gB498-505-tetramers. The highest frequencies of CD8+/H-2Kb/gB498-505+ T cells were measured in HSV-1 KOS-infected mice (17?48±4?62 % among
CD8+ PBL) and the lowest in T0-GFP-infected mice
(6?59±3?59) (Fig. 4c). The frequencies declined to about
2?5 % among CD8+ PBL in wt-infected mice and to 0?6 %
in T0-GFP-infected mice. Thus, infection with wt virus
induces stronger Ab and CD8+ T-cell responses than
infection with the replication-defective virus. Thirty-one
days after infection, all mice were vaccinated with T0HOVA. Seven days later, OVA-specific CTL expansion was
monitored by Tet, as shown in Fig. 2(a). In formerly
seronegative mice (PBS), the frequency of CD8+/H-2Kb/
OVA257-264+ T cells was between 1?29 and 3?14 % among
CD8+ PBLs (Fig. 4d), which corresponded to an approximate increase of specific CD8+ T cells of 10- to 30-fold
compared to naive non-immunized mice (data not shown
and Fig. 2). In contrast, in mice with pre-existing HSV-1
immunity, this frequency of OVA-specific CD8+ T cells
increased only about 3- to 10-fold.
In accordance with reduced expansion of CD8+ T cells in
pre-immune mice (Fig. 4d), a corresponding inhibition
of CTL-effector function could also be detected (Fig. 4e).
Using a novel in vivo killer assay, we tested the ability of
the induced OVA-specific CTLs in the various mice to
directly lyse OVA+ target cells without in vitro restimulation of the T cells (Kleindienst et al., 2005). While the
specific lysis in the control group (non-infected, vaccinated)
was 55 % (Fig. 4e), the differentially pre-infected mice were
only able to develop weak CTL responses [KOS, 5 %; HSV-F,
5?5 %; T0-GFP, 13 % (Fig. 4e)].
These data clearly demonstrate that despite the inherent
immune evasion strategies of HSV-1, infection with wt
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Fig. 4. HSV-1 wt infection reduces vaccine efficiency. C57BL/6 mice were infected by the i.v. route with 26106 HSV-1
KOS, 26106 HSV-1 strain F or 26106 T0-GFP. The control group received 200 ml PBS by the i.v. route. After 31 days, all
mice were vaccinated by the i.v. route with 46106 T0H-OVA. (a) shows an overview of the experimental set-up and timing
(Ab, analysis of serum antibodies; Tet, analysis of Ag-specific Tet+ CD8 T cells from PBL). (b) Sera were taken at days 14
and 26 after infection, diluted 1 : 150 and analysed by ELISA for the presence of anti-HSV-1 IgG (ELISA plates were coated
with HSV-1 MacIntyre Ag, see Methods). (c) The frequencies of HSV-1 glycoprotein B-specific CD8+ T cells in peripheral
blood were monitored 6 and 29 days after infection using H-2Kb/gB498–505-tetramers-PE and anti-CD8-APC mAb for flow
cytometric analyses. (d) At 38 days after infection (=7 days after vaccination) peripheral blood cells were stained with antiCD8-APC and H-2Kb/OVA257–264tetramers-PE and analysed by flow cytometry in order to determine the frequency of
OVA257–264-specific CD8+ T cells. Data represent the mean±SD obtained from five animals per group at each time point.
(e) At 195 days post-infection, five mice of each group were injected with a 1 : 1 mixture of CFSElow-labelled unloaded spleen
cells and CFSEhigh-labelled spleen cells loaded with OVA. After an additional 20 h, mice were sacrificed and spleen cells
analysed for CFSE-positive cells by flow cytometry (‘in vivo killer assay’, see Methods). The FACS results (data not shown)
are displayed as percentage specific lysis. The specific lysis was calculated as described in Methods.
HSV-1 induces strong antiviral immune responses and
interferes significantly (P<0?005 KOS versus PBS and
P<0?005 strain F versus PBS; Fig. 4e) with the efficacy of
a replication-defective, recombinant HSV-1 vaccine vector.
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In addition, our data demonstrate that pre-existing HSVspecific immunity interferes in a similarly effective manner
with subsequently applied analogous vaccines, even if it
has been elicited by replication-competent HSV strains
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HSV-based vaccines in HSV-seropositive hosts
(HSV-F and HSV-KOS) or replication-defective HSV-based
viral vectors (T0-GFP) (P>0?05 KOS versus T0-GFP and
P>0?05 strain F versus T0-GFP; Fig. 4e).
DISCUSSION
Recombinant viruses seem to be promising tools to develop
new-generation vaccines and gene-therapy vectors, but also
confront scientists with several problems (Thomas et al.,
2003). Besides safety issues, one major concern about their
use is the possible impact of pre-existing antiviral immunity
on the vaccine or on gene-therapy efficiency. Especially with
regard to the high prevalence of HSV-1 infection among
the human population, this aspect has to be investigated
carefully for HSV-1-derived vaccine vectors.
Recently, we demonstrated that systemic (i.v.) injection of
a recombinant, replication-defective HSV-1 mutant induces
strong CD8+ T-cell responses that provide protection
against infection with a recombinant intracellular bacterium, L. monocytogenes (Lauterbach et al., 2004). The partly
contradictory results in the literature concerning the influence of pre-existing host immunity on vector efficiency
(Brockman & Knipe, 2002; Chahlavi et al., 1999; Herrlinger
et al., 1998; Hocknell et al., 2002) led us to investigate this
question for the recombinant vector described previously
(Lauterbach et al., 2004). We could demonstrate clearly that
pre-existing anti-HSV immunity reduced humoral (Fig. 1)
and cellular (Figs 2, 3 and 4) immune responses against the
vaccine-encoded Ag.
Vaccination with a replication-defective vhs2 HSV-1
mutant strain has been shown to elicit strong antiviral
IgG responses and to protect mice against challenge with
wt HSV-1 (Geiss et al., 2000). Thus, in order to induce
seroconversion, we first infected mice with a homologous
replication-defective HSV-1 strain. We could show that the
strength of anti-viral Ab responses was highly dependent on
the injection route (Figs 1b, e and 3b). However, even low
anti-HSV IgG titres completely abolished anti-OVA IgG
responses after immunization with T0H-OVA (Fig. 1c) or
reduced them about three- to sixfold (Fig. 1e). Since we
had previously shown that T0H-OVA is a relatively weak
inducer of OVA-specific Ab responses (Lauterbach et al.,
2004), these results were not unexpected. A further reduction of vaccine ‘units’ by pre-existing antiviral immune
responses probably leads to an insufficient amount of OVA
production, which is essential for inducing humoral responses, because OVA protein is not a structural part of
the virus particle itself. Similar results were obtained in a
HSV-amplicon study, in which HIV Env-specific humoral
immune responses were undetectable in pre-infected mice
(Hocknell et al., 2002).
Since our vaccine vector was mainly designed for the induction of strong CD8+ T-cell responses, the results shown in
Figs 2–4 seem to be of greater importance. The expansion
(Figs 2 and 4d) and CTL effector function (Figs 3a, and 4e)
of OVA-specific CD8+ T cells were largely diminished in
http://vir.sgmjournals.org
HSV-seropositive mice. The inhibition of functional CTL
responses could be correlated with the magnitude of
anti-HSV IgG titres (Fig. 3b). These observations are in
accordance with previous studies that demonstrate a
dominant role for immune serum in mediating prophylactic protection against HSV-1 infection (Keadle et al.,
2002). The in vivo assay used as readout in Fig. 3 measures
the rise of urine glucose concentration as a consequence
of fully activated TCR-transgenic CD8+ T cells that lyse
OVA257-264-presenting b-islet cells in RIP-OVAlo mice. The
failure to induce diabetes in pre-infected mice with high
antiviral IgG titres (Fig. 3) does not rule out a partial
activation and thus expansion of OVA257-264-reactive T cells,
as shown in Figs 2(b) and 4(d) for endogenous CD8+Tet+
T cells. These data are in accordance with the findings of
Hocknell et al. (2002) who described a 40–60 % reduction
of cellular immune responses in pre-infected mice after in
vitro restimulation. The stronger inhibitory effects in our
experiments might reflect the differences of in vivo versus in
vitro assays. It is most probable that the low number of
CD8+Tet+ T cells detected after vaccination of pre-infected
animals (Figs 2b and 4d) would show effector functions
after in vitro restimulation. However, when we tested the
effector functions directly in vivo (Fig. 4e), the 90 % reduction in CTL activity observed in HSV-F or HSV-KOS
pre-infected mice reflected a grade of inhibition close to
complete vaccine neutralization (Fig. 4e).
An ICP82 HSV-1 strain encoding b-galactosidase (HD-2
virus) was shown to induce similar Ag-specific IgG responses whether injected into naive or HSV-1-seropositive
mice (Brockman & Knipe, 2002). The discrepancy between
these and our results probably reflects the differential grade
of genetic crippling of the two vaccine vectors: ICP42,
ICP222, ICP272 and vhs2 (this study) versus ICP82 only
(Brockman & Knipe, 2002), respectively. ICP82 mutants
still synthesize the whole spectrum of HSV gene products
that are expressed independently of virus replication, but
infected cells do not produce new virus (Littler et al., 1983;
Morrison & Knipe, 1994; Wu et al., 1988). Thus, this vector
still has the means to modify cell metabolism in favour of
its own gene expression. This could be achieved, among
other strategies, through blocking of RNA splicing by ICP27
(Hardy & Sandri-Goldin, 1994), sustaining protein synthesis through ICP34?5 (Cassady et al., 1998), or the inactivation of CTLs (Sloan et al., 2003) and blocking of
apoptosis (Ogg et al., 2004) by the US3 kinase. In cells
infected with a mutant lacking the immediate-early genes
ICP4, ICP22 and ICP27, only production of ICP6, ICP0,
ICP47 (which is not effective in rodents) and OrfP is to
be expected. Furthermore, the model-Ag b-galactosidase
in the HD-2 virus was fused to the N terminus of a truncated ICP8 (Brockman & Knipe, 2002). Thus, the Ag is
expressed as an integral part of the virus genome, whereas
the Ag in our vector was driven by the hCMV promoter.
The Ag expression might therefore be higher in HD-2infected cells than in T0H-OVA-infected cells. This,
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2407
H. Lauterbach and others
however, remains speculative, because the vaccines were
not directly compared.
Taken together, our data could suggest that an HSV-1-based
vaccine vector that is adapted for high safety (ICP42,
ICP222, ICP272 and vhs2) also shows a higher sensitivity
towards inhibition by pre-existing host immunity, whether
induced by prior immunization or natural infection. With
regard to future applications, it has to be remarked that
Ag-specific CD8+ T-cell responses were inhibited but not
fully abolished (Figs 2b and 4d, e; Hocknell et al., 2002). The
OVA-specific CD8+ T-cell expansion in T0-GFP-treated
mice was not stronger than that of wt HSV-1 pre-infected
mice (Fig. 4d) that had higher HSV-specific IgG levels
(Fig. 4b) and higher frequencies of HSVgB-reactive CD8+
T cells (Fig. 4c). Similarly, the observed differences in CTL
effector function between all pre-infected groups were not
statistically significant (P>0?05).
Thus, after the adaptive immune response has reached a
certain level of protection against HSV, it apparently cannot
be boosted further. This demonstrates nicely the limited
effectiveness of the immune system against infection with
HSV-1, so that even replication-defective HSV-1 mutants
(and also HSV-1 amplicons; Hocknell et al., 2002) still
have the potential to infect cells in an immune animal, even
though to a much lesser extent. Accordingly, in several
vaccine studies in rodents, both the replication of virulent
challenge HSV-1 and disease severity were decreased, but
infection per se was not prevented (Geiss et al., 2000;
Morrison & Knipe, 1994). The mouse immune system is
probably sufficient to keep the virus in check when few cells
are infected. In humans, however, the natural host of
HSV-1, the highly adapted virus might escape the adaptive
immune response through the action of its ‘immuneevasion’ genes, so that even relatively low amounts of virus
can cause disease. This is in accordance with the fact that
so far no anti-HSV vaccine candidate has been shown to be
efficacious in clinical trials [reviewed by Deshpande et al.
(2000)]. Besides, in humans, HSV-specific Ig titres are
only high directly after infection or reinfection [reviewed
by Koelle & Corey (2003)].
What is very unfortunate on the one hand, namely the
failure to vaccinate against HSV, offers on the other hand
the possibility to use HSV-derived vectors for gene therapy
or vaccination, despite the high prevalence of this virus
in the human population. Of course, we are aware of the
limitation of the mouse model in the case of HSV-1, but in
summary, all data argue for a reduced but not abolished
immune response after vaccination with a recombinant
HSV-1 vector. Thus, an immunization protocol with several
injections or a heterologous prime-boost protocol, as shown
by Wang et al. (2003), might provide promising options
for safe HSV-1-based vaccines. In addition, efforts should
be made in the future to optimize HSV-1-vector backbones
in a way such that safety issues do not completely attenuate
the intrinsic immune-evasion mechanisms of HSV-1. This
last feature might turn out to be particularly advantageous
2408
to render HSV-1-based vaccines more efficient in the cases
of pre-existing immunity and of serial applications.
ACKNOWLEDGEMENTS
This work was supported by a grant from the European Commission (EUROAMP; contract QLK2-CT-1999-00055). T. B. was
supported by grants SFB456 and SFB571 from the Deutsche
Forschungsgemeinschaft. P. M. was supported by Italian grants from
the Istituto Superiore di Sanità (ICAV, Italian Concerted Action
on HIV-AIDS Vaccine Development) and from AIRC (Associazione
Italiana per la Ricerca sul Cancro). We thank A. Bol and W. Mertl for
expert help in the animal facility of the Ludwig Maximilians University.
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