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The Journal of Immunology, 2001, 166: 3612-3621.
Copyright © 2001 by The American Association of Immunologists

Antigen-Independent Suppression of the Allergic Immune Response to Bee Venom Phospholipase A2 by DNA Vaccination in CBA/J Mice1

Samantha Jilek, Catherine Barbey, François Spertini and Blaise Corthésy2

Division of Immunology and Allergy, R & D Laboratory, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phospholipase A2 (PLA2) is one of the major honey bee venom allergens for humans. To assess the long-term prevention of allergic reactions by DNA vaccination, a PLA2-CBA/J mouse model was employed using empty or PLA2 sequence-carrying DNA plasmids. Early skin application of either DNA construct before (prophylactic approach) or after (therapeutic approach) sensitization with PLA2/alum led to reduced PLA2-specific IgE and IgG1 titers at 7 mo, with concomitant rise in IgG2a and IgG3. Splenocytes recovered at 5–6 mo after the last DNA administration exhibited a sustained IFN-{gamma} and IL-10 secretion and reduced IL-4 production. Recall challenge with PLA2 boosted IFN-{gamma} and IL-10 secretion, suggesting the reactivation of quiescent memory Th1 lymphocytes. Mice from the prophylactic groups were fully protected against anaphylaxis, whereas 65% of the animals recovered in the therapeutic groups. Th1-polarized immune responses were also active in mice vaccinated with an empty plasmid 32 wk before sensitization with another Ag (OVA). This is the first demonstration that the Ag-coding sequence in DNA vaccine is not necessary to promote immune modulation in naive and sensitized animals for a prolonged period, and has relevance for the understanding of the innate and induced mechanisms underlying gene immunotherapy in long-term treatment of allergy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Specific Ag immunotherapy (SIT)3 represents the preferential treatment of IgE-mediated allergy by regular injections of increasing doses of the allergen over 2–3 years and is effective in preventing subsequent anaphylaxis in people at risk (1). However, the mechanisms underlying the process are not fully understood. In the human honeybee sting allergy, successful therapy is associated with stimulation of venom serum-specific IgG4 and decreasing titers of serum venom-specific IgE (2), the immunologic mediator of anaphylaxis. However, there is no consistent correlation between the occurrence or severity of the reaction to the sting and the titer of serum Ag-specific IgE. Thus, there appears to be undefined factors that mediate the allergic reaction in addition to IgE (3). SIT is a burdensome procedure requiring 3–5 years of treatment and has to be conducted under medical supervision because it causes undesirable side effects in 15% of patients (4). In addition to modulating Ig production, SIT operates by decreasing both Ag-specific T cell proliferation (5, 6, 7) and IL-4 secretion (8), as well as triggering the prototype Th1 cytokine IFN-{gamma} (9, 10).

In addition to immunotherapy with short synthetic peptides (11), large recombinant allergen fragment (12), and long synthetic peptides (9, 10), gene vaccination appears as a safe and inexpensive alternative to classical immunotherapy (13). Recent findings by several laboratories have shown that immunization with DNA vectors encoding human allergens triggers a strong Th1 response to these allergens in rats and mice (14, 15, 16, 17). This resulted in the down-regulation of allergen-specific IgE and IgG1 Ab production, concomitant with an increase in Ag-specific IgG2a titers, and was shown to be mediated by CD4+ T cells. However, these studies did not address in the same experimental model the potential of DNA treatment in a therapeutic setting, the Th2 to Th1 bias at the level of spleen T lymphocytes, the immunomodulatory role of the empty DNA vector before sensitization with the allergen protein, and the long-term (>6 mo) effects of DNA vaccination.

To examine these various aspects, we investigated the mechanisms of immune modulation to bee venom phospholipase A2 (PLA2) in CBA/J mice, which were vaccinated with either DNA constructs containing PLA2 sequences or the empty DNA expression vector. A prolonged time-course analysis conducted over 6 mo revealed that the anti-PLA2 IgE response was suppressed in the prophylactic and therapeutic approaches in an Ag-independent fashion with both empty DNA as well as the DNA vectors carrying PLA2-coding sequences working efficaciously. In addition, PLA2-specific IgG2a and IgG3 titers were increased, marking at the Ab level the expected bias or switch to a Th1 response. This new Ab balance observed 5–6 mo after the last DNA treatment was due to a marked increase of the IFN-{gamma}:IL-4 ratio, as examined using spleen cells cultured in vitro, and was coupled with induction of the suppressive cytokine IL-10. Upon i.p. challenge with native PLA2 performed at 6 mo, spleen cells from vaccinated mice secreted two times more IFN-{gamma} and IL-10 than nonchallenged mice, arguing for active suppression and long-term memory. Mice challenged with otherwise lethal doses of native PLA2 5–6 mo after the last DNA immunization were protected against anaphylaxis, confirming the physiological relevance of the effects analyzed at the cellular and molecular levels. DNA vaccination-induced reduction of Th2-mediated responses was also observed when OVA/alum as the Ag. This indicates that inhibition of primary and recall Ag-specific Th2 cell-mediated responses occurs through the dominant effect of the Th1 background. Together, our data demonstrate that it is possible to successfully select for memory Ag-specific Th cells exhibiting a prevalent Th1 phenotype using independent administration of DNA and Ag.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of expression vectors

The expression vector used was pSecTagA (Invitrogen, Groningen, The Netherlands). Among other features, the vector contains a CMV promoter, an Ig {gamma}-chain sequence for protein secretion, and a polyadenylation site. Expression vectors pSecTagA-PLA2 (PLA2V), pSecTagA-P1 (P1V), pSecTagA-P2 (P2V), and pSecTagA-P3 (P3V) were constructed from pSecTagA (EV) and are coding for the whole bee venom PLA2 and three derived peptides (aa 1–60, aa 47–99, aa 90–134). PCR amplification of the coding regions was conducted using oligonucleotides 1–8 listed in Table IGo and a PLA2 cDNA clone as a matrix (a gift from Mireille Astori, University of Lausanne, Lausanne, Switzerland). The FLAG octapeptide used for detection of the secreted proteins and peptides was inserted at the amino terminus using a second PCR amplification and oligonucleotides 5–8 and PLA2-FLAG (Table IGo).


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  		Table I. Sequences of 5' sense and 3' antisense primers used for cloning1

 
Vector pSecTagA-LacZ coding for Escherichia coli {beta}-galactosidase was generated to confirm the efficacy of the expression vector after intradermal DNA vaccination. The {beta}-galactosidase-coding region was recovered from vector LacZ-EF (a gift from Peter Kao, Stanford University, Palo Alto, CA) using digestion with SalI, Klenow fill-in, and XbaI cut, and then cloned into pSecTagA previously digested with EcoRV and XbaI.

DNA preparation

Different DNA batches amplified at different times show heterogeneous properties in terms of the amount of DNA transcribed and subsequent protein synthesis (18). To decrease the possible effects of these fluctuations on DNA vaccination, each expression vector was amplified independently several times and eventually combined in one unique batch. To get rid of bacterial endotoxin, whose effect on DNA vaccination in a tolerogenic setting can be deleterious, DNA was purified with Triton X-114 (Sigma, Buchs, Switzerland) according to the procedure of Aida and Pabst (19). Before use, DNA was dissolved at a concentration of 5 µg/µl in sterile endotoxin-free water and stored at -20°C.

Mouse DNA vaccination and Ag sensitization

Female CBA/J (H-2k) mice were obtained from Harlan (AD Horst, The Netherlands) and reared in the animal facility in agreement with procedures submitted to the State Veterinary Office. In the prophylactic approach (Fig. 1GoA), mice were vaccinated at 8–10 wk of age with 100 µg of DNA in 20 µl of water once a week for 3 consecutive wk. DNA was applied by pricking a shaved skin area at the base of the tail with a polymethacrylate skin test applicator (Stallergenes, Antony, France). Two weeks later, mice were sensitized with 6 doses of 0.1 µg of PLA2 (Latoxan, Rosans, France) combined with 1 mg of alum (kindly provided by G. del Giudice, Chiron-Vaccines, Siena, Italy) given at 2-wk intervals (20). This indeed favored the induction of an anti-PLA2 IgE response and thus implied that the animals were sensitized with respect to the PLA2 Ag (21). Over a period of 6 mo, mice were bled every 2 wk and sera were conserved at -80°C until further analysis. Mice sensitized with OVA received three to five doses of 1 µg of OVA combined with 1 mg of alum at 1-wk intervals.



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  		FIGURE 1. Experimental setting for the prophylactic and therapeutic approaches used. Arrows indicate the week at which mice were vaccinated with DNA, sensitized with PLA2/alum or OVA/alum, and challenged with PLA2. In the prophylactic approach (A), PLA2-specific Ab were measured from weeks 5–29, whereas in the therapeutic approach (B), Ab were measured from weeks 1–33, with the exception of week 15. Triggering of anaphylaxis, cytokine secretion, and lymphocyte proliferation were evaluated 2 wk after the last Ab titer measurement (PLA2) or 1 wk after the last OVA injection.

 
In the therapeutic approach (Fig. 1GoB), 8- to 10-wk-old mice were first sensitized with six doses of 0.1 µg of PLA2 adsorbed to 1 mg of alum at 2-wk intervals, then vaccinated with DNA constructs as for the prophylactic approach. Sera were collected at 2-wk intervals over 7 mo and kept at -80°C before analysis.

To ensure that DNA vectors were indeed functional, we performed the following control experiment: 2 wk after administration of pSecTagA-LacZ under the same conditions as those used for PLA2 DNA constructs, the {beta}-galactosidase substrate (Roche Molecular Biochemicals, Rotkreuz, Switzerland) applied locally yielded skin blue staining within 1 h, which was completely absent in mice not treated with DNA.

Measurement of serum Ab titers

Ab titers recovered at 2-wk intervals were measured by ELISA. The 96-well Nunc Maxisorp immunoplates (Life Technologies, Basel, Switzerland) were coated for 1 h at 37°C with 50 µl of 5 µg/ml PLA2 (Latoxan) in coating solution (50 mM carbonate-bicarbonate (pH 9.6)). Nonspecific binding sites were blocked with 200 µl of PBS-0.05% Tween 20 (PBS-T)-1% BSA (Fluka, Buchs, Switzerland) and incubated for 1 h at 37°C. After three washes with 300 µl of PBS-T, 50 µl of serial dilutions of mouse serum in PBS-T-1% BSA was added and the plates were incubated overnight at 4°C. After washing as above, 50 µl of detection Ab in PBS-T-1% BSA, namely, 1) biotinylated goat Ab anti-mouse IgG diluted 1:3000 (Caltag, WBAG Resources, Zurich, Switzerland), 2) biotinylated goat Ab anti-mouse IgG1 diluted 1:3000 (Caltag), 3) biotinylated goat Ab anti-mouse IgG2a diluted 1:3000 (Caltag),4) biotinylated goat Ab anti-mouse IgG3 diluted 1:3000 (Caltag), 5) biotinylated rat Ab anti-mouse IgE diluted 1:250 (PharMingen, San Diego, CA) were added to the appropriate wells and incubated for 1 h at 37°C. Plates were washed with PBS-T, and the Ab sandwich was revealed using 50 µl/well of extravidin-alkaline phosphatase (Sigma) diluted 1:10,000. After incubation for 30 min at 37°C, the plates were washed six times with PBS-T before adding 50 µl/well of alkaline phosphatase substrate (1 M diethanolamine (Merck, Zurich, Switzerland), 1 mM MgCl2 and 1 mg/ml p-nitrophenylphosphate (Sigma)). Absorbance values were read at 405 nm and the Ab titers were determined as the reciprocal of the last dilution yielding absorbance values 2-fold higher than the preimmune serum.

PLA2 purification and detoxification for cell culture

To get rid of its intrinsic cytotoxicity on cell cultures, PLA2 in PBS was treated overnight at 37°C with a 100-fold molar excess of DTT (Fluka), then alkylated with a 1000-fold molar excess of N-ethylmaleimide (Fluka). After chemical modification, PLA2 was desalted on a Sephadex G-25 (Amersham Pharmacia Biotech, Zurich, Switzerland) column (1cm x 30 cm) equilibrated and run in PBS.

Lymphocyte recovery, culture, and proliferation assay

Six months after the last sensitization with PLA2/alum (prophylactic protocol) or 5 mo after the last DNA administration (therapeutic protocol), mice were either directly sacrificed or challenged twice with 30 µg of native PLA2 and sacrificed 1 wk later. To assess the response against OVA, mice resistant to PLA2 were sensitized with OVA/alum and sacrificed 1 wk later. Spleen cells from individual animals were plated in a flat-bottom 96-well plate (Costar’ Integra-Biosciences, Wallisellen, Switzerland) at 15–20 x 104 cells per 200 µl of DMEM (Life Technologies) complemented with 10% FCS (Life Technologies), 20 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies) and 5 x 10-5 M 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies). Alkylated PLA2 (10 µg/ml; see above) resuspended in plain DMEM was added for Ag-specific proliferation. Con A (Sigma) used at 2.5 µg/ml served as positive control. OVA used at 10 µg/ml and medium alone were used as negative controls. The cells were incubated for 4 days at 37°C and finally pulsed overnight with 1 µCi/well [methyl-[3H]thymidine (Hartmann Analytic, Braunschweig, Germany). Cells were then harvested and nuclear incorporation of radioactivity was measured in a scintillation beta counter (Topcount; Canberra Packard, Zurich, Switzerland). Proliferation responses were calculated as stimulation index dividing geometric mean Ag-stimulated cpm by background cpm.

Cytokine release assays

One million cells were incubated in 24-well plates in a final volume of 1 ml in the presence of PLA2, Con A, OVA, or plain medium. Supernatants were harvested at the indicated times, and cytokine concentrations were determined by ELISA using a combination of specific mAb according to the manufacturer’s protocol: IL-4: PharMingen, clones 11B11 and BVD6-24G2, 3-day-old supernatant; IFN-{gamma}: PharMingen, clones R4-6A2 and XnG1.2, 2-day-old supernatant; and IL-10: PharMingen, clones JES5-2A5 and SXC-1, 3-day-old supernatant.

Statistical analysis

Comparison in cytokine secretion and T cell proliferation assays between groups of mice (see Figs. 3Go, 5Go, and 6Go) was evaluated by the paired Student t test using GraphPad Instat software Mac version 2.01 (San Diego, CA). SDs of Ab titers (see Figs. 2Go and 4Go) were calculated using the function STDEVA from the Excel 98 application for Apple Macintosh (Cupertino, CA).



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  		FIGURE 3. Long-term analysis of immune markers of the DNA-vaccinated mice (prophylactic groups). A, Analysis of the cytokine production of splenocytes recovered from mice prophylactically vaccinated with various DNA constructs and sensitized with six i.p. injections of PLA2/alum. U, Untreated, sensitized mice. Mice were challenged with PLA2 ({blacksquare}) at week 31 (recall challenge) or left unchallenged ({square}). Splenocytes were incubated with the indicated Ag and cytokines were measured as described in Materials and Methods. B, Stimulatory index (S. I.) of splenocytes from mice challenged with PLA2 ({blacksquare}) at week 31 or left unchallenged ({square}). Proliferation was performed in the presence of 10 µg/ml detoxified PLA2 for 5 days. Splenocytes from untreated, nonsensitized mice yielded background levels (data not shown). C, Lack of anaphylactic reaction of vaccinated mice. All control mice (-) given 30 µg of PLA2 i.p. died of anaphylactic shock, whereas mice treated with any DNA construct rapidly recovered from two identical PLA2 doses.

 


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  		FIGURE 5. Long-term analysis of immune markers of the DNA-vaccinated mice (therapeutic groups). A, Analysis of the cytokine production of splenocytes recovered from mice therapeutically vaccinated with various DNA constructs after sensitization with six i.p. injections of PLA2/alum. U, Untreated, sensitized mice. Mice were challenged with PLA2 ({blacksquare}) at week 35 (recall challenge) or left unchallenged ({square}). Splenocytes were incubated with the indicated Ag and cytokines were measured as described in Materials and Methods. B, Stimulatory index (S. I.) of splenocytes from mice challenged with PLA2 ({blacksquare}) at week 35 or left unchallenged ({square}). Proliferation was performed in the presence of 10 µg/ml detoxified PLA2 for 5 days. Splenocytes from nonsensitized, untreated mice yielded background levels (data not shown). C, Lack of anaphylactic reaction of vaccinated mice. All control mice (-) given 30 µg of PLA2 i.p. died of anaphylactic shock, a phenomenon limited to one-third of the mice treated with any DNA construct challenged with one or two PLA2 doses.

 


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  		FIGURE 6. DNA vaccination protects mice against another irrelevant Ag. A, Mice were vaccinated with pSecTag (EV), then sensitized with OVA, and OVA-specific IgE, IgG2a, and IgG3 titers were examined 14 wk after the last DNA application. Untreated mice (U) given PBS before OVA sensitization served as controls. B, Splenocytes of vaccinated or untreated mice in A were cultured in vitro for 2–3 days in the presence of OVA. Secretion of IFN-{gamma} and IL-4 was measured by ELISA. C, Mice vaccinated with EV in the prophylactic group were sensitized with OVA at weeks 35, 36, and 38. The OVA-specific IgE titer was compared with that of untreated mice (U) sensitized under the same conditions. D, Splenocytes of animals used in C were cultured in vitro in the presence of OVA (O) or plain medium (M). Cytokine secretion was measured in the supernatant after 2–3 days in culture (left panel) and proliferation was assessed at 5 days (right panel).

 


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  		FIGURE 2. Modulation of total IgG, IgG1, IgG2a, IgG3, and IgE PLA2-specific response of CBA/J mice, which had been inoculated with DNA once a week for 3 wk before Ag sensitization (prophylactic approach). Results are expressed as serum titers (IgG isotypes) or OD units using 1:40 dilutions (IgE). IgG and IgE levels in untreated mice remained stable for the whole course of the experiment. Sera were assayed from weeks 5–29 and at week 32 after PLA2 challenge. Data were averaged from five mice per group and are expressed as mean ± SD. EV, empty expression vector; PLA2V, expression vector containing the whole PLA2-coding sequence; P1V, expression vector coding for aa 1–60 of PLA2; P2V, expression vector coding for aa 47–99 of PLA2; P3V, expression vector coding for aa 90–134 of PLA2.

 


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  		FIGURE 4. Modulation of total IgG, IgG1, IgG2a, IgG3, and IgE PLA2-specific response of CBA/J mice, which had been inoculated with DNA once a week for 3 wk after initial Ag sensitization (therapeutic approach). Results are expressed as serum titers (IgG isotypes) or OD units using 1:40 dilutions (IgE). Sera were assayed from weeks 1–13, then from weeks 17–33, and finally at week 36 after PLA2 challenge. In untreated mice, IgG and IgE titers remained stable for the duration of the experiment. Data were averaged from five mice per group and are expressed as mean ± SD. Plasmid nomenclature corresponds to that in Fig. 2Go.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA vectors coding for PLA2 and peptides are functional in vitro and in vivo

After cloning as described in Materials and Methods, Chinese hamster ovary cells were transfected with either one of the four expression vectors coding for the full-length PLA2 and derived peptides, and the production of proteins was assessed by dot blot and immunofluorescence (S. Jilek, unpublished observations). Proteins were found both intracellularly and in the culture medium, indicating that the vectors are able to govern transcription and translation in eukaryotic cells. As an additional control, pSecTagA-LacZ was administrated to CBA/J mice in the form of three doses of 100 µg at 1-wk intervals. Two weeks after the last application, the presence of the {beta}-galactosidase enzyme at the site of administration was determined by injecting s.c. 150 µl of the substrate. Within 1 h, the skin of animals exposed to pSecTagA-LacZ turned blue, whereas control animals did not show any change in the skin complexion (S. Jilek, unpublished observations). With time, the staining was seen not only in the application zone but also in surrounding areas, indicating that the protein expressed by the cells at the site of injection diffused in the extracellular milieu. It took 24 h after substrate injection to observe complete disappearance of the coloration. Crucial to the subsequent interpretation of the data, these results indicate that all of the expression vectors are fully active in both in vitro and in vivo contexts.

PLA2-specific IgG1, IgG2a, IgG3, and IgE responses are modulated by prophylactic DNA vaccination

Either PLA2 or 45–60-aa-long peptides derived from PLA2 down-regulate specific IgE response in mice when given i.p. or via the nasal route (9, 10). However, relatively large amounts need to be administered repeatedly. To explore the potential of DNA vaccination to modulate the allergic immune response and prevent anaphylaxis, plasmids coding for PLA2, the PLA2-derived peptides devoid of possible recognition by IgE, and the empty vector were transfected via intradermal vaccination using three doses of 100 µg given at 1-wk intervals. Animals were then sensitized with 6 doses of PLA2/alum (see Materials and Methods), and the immunomodulation was analyzed first by measuring changes of the PLA2-specific Ig titers over a 6-mo time course (Fig. 2Go). Surprisingly, all DNA constructs containing or lacking PLA2-coding sequences led to very similar results. Prophylactic DNA treatment initially increased allergen-specific IgE production, as previously observed in conventional immunotherapy (22). Starting at week 21, gene vaccination reduced IgE responses against PLA2 Ag for the duration of the analysis, in contrast to untreated animals exhibiting stable titers twice as high as those measured at week 17 (10; B. Corthésy, unpublished observations). The last measurement yielded average IgE titers even below those observed at the end of the sensitization phase. Consistent with the results, the weak burst seen for specific IgG1 Ab titers started decreasing 6 wk after sensitization, reflecting the inability of the vaccinated mouse to sustain production of this Th2-type Ig. An increase in specific IgG2a was obtained following PLA2/alum sensitization, suggesting a DNA vaccine-mediated skew toward a Th1 type of immune response (23). The rise in IgG2a titer seen with construct P1V known to contain a dominant T epitope (9) was largely due to two animals and was not reproduced using the PLA2V construct. When extending the analysis to the IgG3 isotype, a sustained titer rise was also detected, therefore suggesting at the Ab level that the immune response was actively biased to Th1 cytokines. Similar to IgE, IgG titers were two to four times higher with no DNA treatment and remained stable for the duration of the analysis. The very similar trend seen on the Ig pattern of either DNA constructs argues in favor of a dominance of the Th1-mediated response that was examined at the cellular and molecular levels.

Prophylactic DNA vaccination triggers a Th1 cytokine shift, modulates specific T cell responsiveness, and prevents anaphylaxis

Immune polarization was examined by the capacity of lymphocytes to secrete more IFN-{gamma} (8) and IL-10 (24). In addition, the Th2 to Th1 switch is concomitantly accompanied by a marked drop in IL-4 synthesis (25, 26). T cell responses were thus assayed by proliferation of spleen cell cultures, since these latter yield the same cytokine profile as lymph node cells (9). T cells from mice immunized with either DNA plasmids recovered 30 wk after the last exposure to PLA2 produced up to 11-fold (p < 0.0005) more IFN-{gamma} and 4.5-fold (p < 0.0008) more IL-10 than spleen cells from untreated, sensitized CBA/J mice kept in the same environment, but sensitized with PLA2/alum (Fig. 3GoA). The production of IL-4 was still detectable in the assay, but significantly reduced (p < 0.001) as compared with that seen using splenocytes isolated from untreated, sensitized mice. Together, this resulted in a pronounced rise in the Th1:Th2 ratio expected after skin surface DNA application. When mice were challenged twice with 30 µg of native PLA2 followed by spleen cell extraction 1 wk later, we observed a 2-fold increase in the amount of IFN-{gamma} and IL-10 (p < 0.005) after PLA2 stimulation in vitro, with negligible changes in the IL-4 concentration (Fig. 3GoA). No triggering of IFN-{gamma} and IL-10 secretion was observed when the spleen cells were cultivated in the presence of medium alone or in the presence of 10 µg/ml OVA, although the three cytokines were still produced to a similar extent as before PLA2 challenge (Fig. 3GoA). Together, the data indicate that DNA vaccination has prompted the T cell response to evolve toward a Th1 profile and that subsequent sensitization with PLA2/alum allows an Ag-specific memory response to be preserved for up to 7 mo. Moreover, after PLA2 challenge, no significant change in the PLA2-specific Ab responses could be measured at week 32 (Fig. 2Go), suggesting Th1-polarized memory responses.

No T cell proliferation could be detected using the spleen of DNA-vaccinated mice left for 7 mo in the absence of any PLA2 challenge (Fig. 3GoB) or the spleen of untreated, nonsensitized mice (S. Jilek, unpublished observations). In contrast, two doses of 30 µg of PLA2 before spleen cell recovery made them capable of proliferating specifically (p < 0.004) in the presence of PLA2 in vitro (Fig. 3GoB). Consistent with the increase in IFN-{gamma} and IL-10 expression, this suggests that an active immune deviation mediated by Th1 cytokines is actually taking place, coupled with a state of unresponsiveness in the absence of Ag challenge. Furthermore, the physiological relevance of 1) the resistance to IgE induction, 2) the production of cytokines of the Th1 type, and 3) the capability to proliferate upon specific Ag stimulation is demonstrated by the 100% survival rate of DNA-vaccinated mice exposed to one and two injections of 30 µg of PLA2 (Fig. 3GoC). The protection against anaphylaxis was dependent on the prophylactic treatment, as sensitized, nonvaccinated mice all underwent an immediate drastic temperature drop and died within 30 min after the first administration of challenging PLA2 (Fig. 3GoC).

Therapeutic DNA vaccination prevents PLA2-specific IgE response, enhances Th1 cytokine secretion, and partially blocks anaphylaxis

Given the high effectiveness of DNA vaccination both in terms of preventing the development of an IgE-mediated response and inducing long-lived protective immune memory, we examined the possibility of down-regulating the course of an established allergic response. Mice were vaccinated intradermally using three doses of 100 µg of DNA constructs given at 1-wk intervals. The same markers of the immune response as for the prophylactic protocol were analyzed. Following PLA2 sensitization, the PLA2-specific Ig Ab titers raised during the first 4 wk with a kinetics lacking the lag phase seen in the prophylactic approach (Fig. 2Go). DNA vaccination led to the reduction of IgE and IgG1, with a concomitant increase of IgG2a and IgG3, yet to a less marked, but appreciable, extent than in mice treated with the prophylactic approach (Fig. 4Go). Mice not treated with the DNA vaccine kept exhibiting stable IgG and IgE titers (Ref. 9; S. Jilek, unpublished observations). Once again, the presence of PLA2-coding sequences on the DNA vector did not significantly affect the fluctuations in Ig titers. This suggests that a pre-established Th2 allergic response can be redirected by Ag-independent DNA therapy favoring secretion of Ig isotypes controlled by Th1 cytokines. Consistent with this, no significant change in the PLA2-specific Ab responses could be measured at week 36 following PLA2 challenge (Fig. 4Go).

T cells recovered from spleen 5 mo after the last exposure to PLA2/alum were found to be able to secrete IFN-{gamma} and IL-10 to a comparable extent as T cells obtained from mice in the prophylactic groups, well above (p < 0.0003) the level detected in untreated, sensitized mice (Fig. 5GoA). Challenge with native PLA2 led to a roughly two times higher release of IFN-{gamma} and IL-10 by PLA2-stimulated T cells (p < 0.008). For the IL-4 production, we observed a 3-fold drop (p < 0.01) as compared with that measured with cells from untreated, sensitized mice, which reflected the observation made at the Ab level. Before PLA2 challenge, no Ag-specific proliferation could be detected, whereas a 2- to 3-fold increase (p < 0.007) in the stimulation index was obtained after PLA2 challenge (Fig. 5GoB). Similar to what we concluded from the prophylactic protocol, PLA2 challenge reactivated quiescent T cells to secrete IFN-{gamma} (27) and maintain a Th1 milieu attenuating the allergy-oriented Th2 immune response. In contrast to the full protection against anaphylaxis seen with mice treated prophylactically, 70% of the mice showed long-lasting immobility, of which half did not recover and eventually died (Fig. 5GoC). Interestingly, mice showing no sign of anaphylaxis after the first challenge could bear a second challenge with 30 µg of PLA2. No significant correlation with the remaining IgE titer could be drawn, a situation also encountered in conventional immunotherapy involving human patients.

The Th1 milieu resulting from DNA vaccination inhibits primary and recall responses against OVA used as a control Ag

Our data have shown that the Ag-coding sequences are not essential in the DNA plasmid during the initial phase of Th2 to Th1 deviation, and that the Th1 milieu itself is sufficient to take control of the allergic reaction induced by Ag administration before or after gene therapy. We therefore reasoned that the unexpected observation we made for the PLA2 protein would benefit from its confirmation using another Ag protein and thus substantiate the dominant Th1 bystander effect revealed in this study. Two series of control experiments were conducted with OVA. First, mice were vaccinated three times with empty pSec-TagA at 1-wk intervals, and 2 wk after the last DNA application were sensitized five times with OVA/alum at 2-wk intervals. OVA-specific IgE measured in the serum of vaccinated mice 2 wk after the last OVA/alum injection were reduced by a factor of 3.5 as compared with untreated, sensitized mice (p < 0.002), while IgG2a and IgG3 levels were augmented by a factor of 1.8 and 2.8 (p < 0.005) (Fig. 6GoA). Similar to the prophylactic approach with the PLA2 Ag, the cytokine secretion was biased to a Th1-type response, with IFN-{gamma} taking over IL-4 as reflected by the significant ratio changes (p < 0.006) of the two cytokines (Fig. 6GoB) and OVA-dependent proliferation (B. Corthésy, unpublished observations). Second, mice subjected to the PLA2 prophylactic protocol were sensitized 6 mo after the last DNA application with three i.p. applications of OVA/alum. When compared with naive mice, the level of OVA-specific IgE Ab molecules was reduced by a factor of 2.6 (p < 0.002) in vaccinated animals (Fig. 6GoC). In addition, DNA-vaccinated mice did not show any sign of anaphylaxis when challenged with OVA. It thus appears that gene therapy maintains a Th1-protective medium whose effect can be detected for long periods of time, even in the context of a different Ag. It appears that a "cytokine milieu memory effect" takes place and is sufficient to produce Th1-biased responses at the time the Ag is administered. Consistent with this observation is the OVA-specific proliferation of spleen T cells recovered 1 wk after the last sensitization with OVA/alum yielding a stimulation index close to 3 (p < 0.001) (Fig. 6GoD). This implies in active suppression long-term DNA-primed, IFN-{gamma}-producing T cells (Fig. 6GoD) having acquired the capacity to respond to OVA after three consecutive injections. Likewise, untreated mice produced IL-4 and responded to OVA proliferation in vitro.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intradermal gene vaccination of mice induces Ag-specific Th1 cells that secrete high levels of IFN-{gamma} and stimulates production of Abs of the IgG2a and IgG3 isotypes (28). Our data show that the effect of DNA immunization is dominant, since it prevents the subsequent induction by PLA2/alum of either an IgE Ab response or activation of Th2 cells producing IL-4; it can also reduce a pre-existing allergen-specific IgE response. Whether this is accompanied by the down-regulation of basophils and mast cell activation by IFN-{gamma} remains to be determined (29, 30). The novelty of our data resides in the observation that the allergen does not need to be codelivered by the DNA plasmid, but its mere presence as an exogenously delivered protein after gene vaccination promotes naive CD4+ T lymphocyte differentiation toward Th1 cells, leading to a second burst of IFN-{gamma} production in an allergen-dependent fashion. When gene vaccination was followed by Ag delivery, reversal of Th2-type responses was observed, underlining the plasticity of the system in vivo and the dominant effect of DNA-induced Th1 cytokines over a pre-existing Th2 pattern. Specifically, we demonstrate herein that PLA2-independent gene vaccination down-regulates an ongoing Th2 response in favor of a Th1 profile in both a prophylactic and therapeutic approach, ultimately resulting in prevention of anaphylaxis in CBA/J mice.

In other studies describing the preventive action of DNA immunotherapy in allergy models, the properties of empty DNA constructs were not evaluated (18) or, if so, were examined in other mouse and rat strains and more importantly after i.m. injection (14, 16, 31), resulting in tissue injury and inflammation favoring stronger local immune response (32). Furthermore, in contrast to our own experimental setting, DNA preparations were not devoid of bacterial endotoxin. It remains to determine whether this is crucial to the outcome of the immune response. DNA plasmids administered intradermally have been followed through in BALB/c mice, and tissue spreading beyond regional lymph nodes has been established (33). However, RT-PCR analysis of various tissues after injection of DNA revealed no Ag expression (34), raising the question of the identity of the APCs involved and the necessity of presentation at the time of DNA "reading" by the host (35). Consistently, our data stress that the cytokine milieu obtained following DNA vaccination is crucial and sufficient to the generation of a Th1-skewed immune response, without excluding that the tiny amounts of Ag produced in vivo (in the pg ml-1 range; 36) might conceptually help to maintain a low level of Ag-specific Ab and/or modulate the cytokine pattern in a dose-dependent fashion (37). Remarkably, the role of the Th1 milieu before Ag challenge is reproduced when using OVA in EV-vaccinated mice. It remains to be established whether Ag of a different nature will conduct to the same observations as those reported in this work. In this respect, birch pollen isoforms coded by DNA plasmids triggered either a strong Ag-specific Th1 response (Betv1a), or resulted in no proliferation nor cytokine release (Betv1d) in the absence of sensitization (38).

Our results point to the fact that immunotherapy can be accomplished by DNA lacking any coding sequences for the allergen under study. What therefore are the possible mechanisms explaining the protective function of Ag-independent gene vaccination? A clue to this puzzling question might come from the concept that DNA used for vaccination can be divided into two units consisting of a transcriptional unit directing Ag synthesis and an adjuvant/mitogen unit in the plasmid backbone acting on cells of the innate immune system (32). The Th1 bias resulting from the adjuvant properties of bacterial DNA has been ascribed to a molecular palindromic motif exhibiting the sequence: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' (39, 40) and referred to as immunostimulatory sequence (ISS). In the absence of cytosine methylation, oligodeoxynucleotides (ODN) containing such motifs promote secretion of IFN-{alpha}{beta}, IL-12, and TNF-{alpha} by macrophages (41), stimulation of dendritic cells (42), and production of IFN-{gamma} by NK cells (43). Thus, the ISS are able to activate cells belonging to the innate branch of the immune system and trigger an initial burst of IFN-{gamma} in an allergen-independent manner.

The pSecTagA used in this study comprises as many as 23 ISS, and thus it makes sense that it can stimulate the innate immune system to create a cytokine milieu that favors the generation of a Th1-biased response to the Ag. The in vivo adjuvant activity of ISS before Ag administration is referred to as "prepriming" (44, 45). Consistent with our observations that ISS per se can suppress markers of an allergic reaction, prevention of allergic lung inflammation in a mouse model of asthma was reduced by intratracheal administration of CpG ODN alone before allergen challenge (46). The CpG ODN increased the ratio of IFN-{gamma}:IL-4, diminished eosinophilia, and reduced Ag-specific IgE-producing cells. Similar to our data, a sustained Th1 memory response to the recall Ag was detected for at least 6 wk after ODN administration (46). This is attributed to increased IFN-{gamma} concentrations and decreased IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluids (47). In the case of allergic hyperresponsiveness, ISS should hence be seen as dominant negative modulators (48) due to their intrinsic capacity to positively influence the development of memory Th1 cells.

Our data show that the plasmid DNA does not need to contain the coding sequence for the allergen to down-regulate the allergic response, implying that Th1 deviation by itself is sufficient to protect against subsequent challenge with the allergen protein. This is indeed reflected by the OVA sensitization of mice, which resulted in the preserved secretion of IFN-{gamma} in the culture supernatant of the same cells in vitro (Fig. 6Go). Likewise, P2V and P3V lacking T epitope in the coding sequences they carry are as good as EV in preventing production of markers of allergic and Th2 immune responses. In contrast to P2 and P3 peptides administered as such (9), IgE Ab titers to OVA injected 6 mo after the last DNA application (EV or PLA2V) were reduced as compared with nonvac-cinated animals; this suggests that the prevalence of the Th1 response was preserved as marked by sustained IFN-{gamma} production and can be seen as what we call a cytokine milieu memory effect. DNA, or ISS thereof, might confer to APC the capacity to present Ag to T cells bathed within a Th1-biased cytokine milieu and therefore prime the synthesis of IgG2a and IgG3 Ab preferentially.

Remarkably, our data demonstrate that skin surface scrapping of DNA plasmids containing ISS motifs rapidly stimulating the host to mount a Th1-dominated innate response is operative in both the prophylactic and therapeutic settings. In addition, the documented absence of local inflammation after intradermal DNA immunization might preclude the induction of costimulatory molecules including CD86, CD40 (49) on APCs (most likely skin Langerhans cells), and maintain these latter in a status of presentation inducing tolerance (50). Another advantage of DNA resides in its capacity to "survive" for long periods in the body (51) and thus function as some sort of an adjuvant reservoir favoring Th1 type cytokines. We believe this might account for the long-term memory seen in this study and also explain the rapid burst in the production of IgG2a and IgG3 directed against sensitizing doses of PLA2 in the prophylactic or therapeutic approaches. Interestingly, the maintenance of IL-4 production suggests that the vaccinated organism is not fully impaired in its potential to mount a Th2-type immune response. This has implications in protection against parasites for example (52).

The increase in PLA2-specific IgG2a and IgG3 Abs observed in mice after prophylactic and therapeutic gene vaccination might block serum-facilitated allergen presentation (53), thus mimicking the protective function of IgG4 in humans (54). The role of IgG1 Abs blocking the Ag-IgE binding through recognition of similar epitopes (55) suggests that the Th1/Th2 dichotomy reflected by production of Ig isotypes might represent an oversimplification when seeking markers of immunomodulation. However, the contribution of various maternal Ag-specific IgG1 and IgG2b Abs to suppression of the IgE immune response to bee venom PLA2 in CBA/J mice offspring argues in favor of the possible role of such Ab (21). The relevance of serum-facilitating allergen presentation is further acknowledged by the recent report of van Neerven et al. (56) who were able to demonstrate its effectiveness in patients allergic to birch pollen.

In summary, this is the first report that Ag-independent and prolonged suppression of an allergic reaction is modulated by DNA vaccination. Intradermal administration of DNA lacking any coding sequence for the Ag preferentially 1) stimulated the production of Th1 cytokines, 2) suppressed Ag-specific IgE, 3) triggered Ag-specific IgG2a and IgG3, 4) and blocked or reduced anaphylaxis after prophylactic and therapeutic treatment, respectively. The data presented in this study should prompt the further examination of DNA vaccination in the context of the expected effects (Th switch, Ab production, cellular responses, induction of anergy) on the immune system in terms of Ag mobilization, the sites and means of delivery, the amount and nature of DNA, as well as the target organisms (31). It is conceivable that the administration of ISS with a given allergen could be used in atopic persons to modify the Th2-oriented allergen-specific response. In addition, in the animal experiments that mirror the situation in humans, it is now possible to analyze whether the deficit in Th1-stimulating microbial infections encountered in developed countries (and suggested as one possible cause for the increase of atopic diseases (57, 58, 59)) might be compensated for by treatment with Ag-independent, ODN-mediated gene therapy.


   Acknowledgments
 
We thank Drs. Eric Bernasconi, William Blanco-Bose, and Alain Sauty for critical reading, comments, and suggestions.


   Footnotes
 
1 This work was supported by Swiss National Science Foundation Grants 3100-050912 and 3200-057088 (to B.C.) and Grant 3100-059482 (to F.S.). Back

2 Address correspondence and reprint requests to Dr. Blaise Corthésy, R & D Laboratory, Division of Immunology and Allergy, Hôpital Orthopédique, Avenue Pierre Decker 4, CH-1005 Lausanne, Switzerland. Back

3 Abbreviations used in this paper: SIT, specific Ag immunotherapy; FLAG, the octapeptide DYKDDDDK; ISS, immunostimulatory sequence; ODN, oligodeoxynucleotide; PLA2, bee venom phospholipase A2; PBS-T, PBS-Tween 20. Back

Received for publication August 17, 2000. Accepted for publication December 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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