Reduced immune responses after vaccination with a recombinant herpes simplex virus type 1 vector in the presence of antiviral immunity

Henning Lauterbach1,{dagger}, Christine Ried1, Alberto L. Epstein2, Peggy Marconi3 and Thomas Brocker1

1 Institute for Immunology, Ludwig Maximilians University Munich, Goethestrasse 31, 80336 Munich, Germany
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

Correspondence
Thomas Brocker
tbrocker{at}med.uni-muenchen.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.

{dagger}Present address: The Scripps Research Institute, Division of Virology, Department of Neuropharmacology, La Jolla, CA, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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; Wade-Martins 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).

Upon HSV-1 infection, innate immunity provides the first line of defence, with activated macrophages, dendritic cells (DC), NK cells and {gamma}{delta} 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 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 HSV-vaccine 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-1-seropostive 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mice.
All mice were bred and maintained under standard conditions 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 {beta}-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, 2x106 p.f.u. HSV-1 KOS or strain F were injected intravenously (1x107 p.f.u. ml–1 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 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 4x106 rHSV-1 (200 µl)–1 for intravenous (i.v.), intraperitoneal (i.p.) and subcutanous (s.c.) injection, and to 4x106 rHSV-1 (50 µl)–1 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, 1x106 transgenic T cells were injected intravenously into recipient mice.

Enzyme-linked immunosorbent assay (ELISA).
For the detection of OVA-specific antibodies, 96-well microtitre plates (Nunc Maxisorp) were coated with 15 µg chicken OVA ml–1 (Sigma Chemicals Co.) at room temperature overnight. For the detection of anti-HSV antibodies, plates were either coated with T0-GFP in PBS (2x105 p.f.u. ml–1) overnight at 4 °C 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, HRP-labelled 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 3,3',5,5' tetramethylbenzidine, 0·3 µl ml–1 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 performed 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 10–6 M OVA257-264-peptide for 1 h at 37 °C, washed and labelled with a high concentration of carboxy-fluorescein diacetate succinimidyl ester (CFSE, 2·5 µM) (CFSEhigh cells). The second control population was labelled with a low concentration of CFSE (0·25 µM) (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 2x107 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 5x105 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=[1–(ratio unprimed/ratio primed)x100].


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 HSV-specific Ab (Fig. 1b, e). Four weeks post-infection with T0-GFP, 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 OVA-specific IgG titres could be detected at 2 weeks after T0H-OVA 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 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 HSV-1-vector-encoded Ag are affected in HSV-1-seropositive animals.



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Fig. 1. Inhibition of Ab induction by pre-existing immunity. C57BL/6 mice were infected at day 0 with 4x106 T0-GFP via the indicated routes (a, d). Four weeks later, mice were immunized with 4x106 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.

 
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 T0H-OVA challenge only 0·63±0·38 % CD8+Tet+ T cells among CD8+ PBLs (Fig. 2b). These data show that pre-existing immunity in HSV-seropositive mice has a negative influence on CD8+ T-cell responses.



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Fig. 2. Inhibition of Ag-specific CTL expansion by pre-existing immunity. C57BL/6 mice were infected with 4x106 T0-GFP by the i.v. route or not treated 21 days before i.p. vaccination with 4x106 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.

 
Inhibition of CTL effector functions in the RIP-OVAlo 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 {beta}-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 Ag-specific 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). RIP-OVAlo mice that had received adoptively transferred OVA-specific CD8+ T cells (OT-1>RIP-OVAlo) were left 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>RIP-OVAlo mice were immunized by the i.v. route with T0H-OVA 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 became diabetic. In mice that had received i.d. or i.p. T0-GFP, 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 T0H-OVA 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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Fig. 3. Inhibition of CTL effector functions in the transgenic RIP-OVAlo mouse model. RIP-OVAlo mice that had received 1x106 adoptively transferred OT-1 CD8+ T cells were infected with 4x106 T0-GFP via the routes indicated or left untreated. All mice were vaccinated 19 days later with 4x106 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).

 
Wt virus infection interferes with vaccine efficiency
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 T0H-OVA. 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.



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Fig. 4. HSV-1 wt infection reduces vaccine efficiency. C57BL/6 mice were infected by the i.v. route with 2x106 HSV-1 KOS, 2x106 HSV-1 strain F or 2x106 T0-GFP. The control group received 200 µl PBS by the i.v. route. After 31 days, all mice were vaccinated by the i.v. route with 4x106 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 anti-CD8-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.

 
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 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. In addition, our data demonstrate that pre-existing HSV-specific immunity interferes in a similarly effective manner with subsequently applied analogous vaccines, even if it has been elicited by replication-competent HSV strains (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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 vhs 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 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 {beta}-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 ICP8 HSV-1 strain encoding {beta}-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: ICP4, ICP22, ICP27 and vhs (this study) versus ICP8 only (Brockman & Knipe, 2002), respectively. ICP8 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 {beta}-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-2-infected cells than in T0H-OVA-infected cells. This, 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 (ICP4, ICP22, ICP27 and vhs) 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 ‘immune-evasion’ 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 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.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 13 April 2005; accepted 14 June 2005.



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