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The Journal of Immunology, 1999, 163: 5913-5919.
Copyright © 1999 by The American Association of Immunologists

A Long-Term Memory Obtained by Genetic Immunization Results in Full Protection from a Mammary Adenocarcinoma Expressing an EBV Gene1

Jehad Charo2,*,{dagger}, Anne-Marie T. Ciupitu*,{dagger}, Alain Le Chevalier de Préville*, Pankaj Trivedi3,{dagger}, George Klein{dagger}, Jorma Hinkula{ddagger} and Rolf Kiessling*,{dagger}

* Cancer Center Karolinska (CCK), Karolinsk Hospital, Stockholm, Sweden; {dagger} Microbiolgy and Tumorbiology Center (MTC), Karolinska Institute, Stockholm, Sweden; and {ddagger} Swedish Institute for Infectious Disease Control, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have tested the capability of a plasmid DNA (pDNA) expressing the EBV nuclear Ag-4 (EBNA-4) to evoke a T cell response-associated protective immune response against a tumor expressing this gene. We have found that ACA mice immunized with EBNA-4-expressing plasmid were partially protected against syngeneic mammary carcinoma line (S6C) expressing EBNA-4 (S6C-E4). This protection was enhanced by coimmunizing mice with EBNA-4- and GM-CSF-expressing plasmids, and a full protection was achieved by coimmunizing mice with EBNA-4- and IFN-{gamma}-expressing plasmids. Furthermore, mice that have rejected the EBNA-4-positive tumor were also resistant against a subsequent challenge with the original nontransfected tumor line. We then checked for the ability of pDNA immunization to provide a protective long-term memory response. We indeed found that even after 3 mo from the last immunization, full protection was obtained by this method, as compared with full tumor outgrowth in the control-immunized group. These findings support the concept that a nonviral, pDNA-based vaccination strategy is useful to fully protect from the outgrowth of tumors expressing this EBV gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epstein-Barr virus is a large dsDNA, {gamma} herpes virus (1, 2, 3). It is the causative agent of infectious mononucleosis, a benign lymphoproliferation disease that could be fatal in patients with X-linked lymphoproliferative syndrome (XLPS).4 The virus is also associated with an increasing number of malignancies including: Burkitt’s lymphoma (BL), nasopharyngeal carcinoma, posttransplant lymphoproliferative disorders (PTLD), leiomyosarcoma, AIDS-associated lymphoma, lymphoepithelioma-like carcinoma, Hodgkin’s disease, peripheral T cell lymphoma, and to a lesser extent with gastric carcinomas and African breast carcinoma (2, 4, 5, 6). The morbidity and mortality associated with EBV make it a good target for the development of new treatments, including preventive and therapeutic vaccines (7, 8). These, however, have been hindered due to the absence of a good experimental animal system because the virus is strictly human specific (1, 2, 5).

Several new principles of treating EBV-associated diseases have been considered (9, 10, 11, 12, 13, 14, 15). Gene therapy approaches using suicide constructs that are specifically or nonspecifically activated in EBV-positive cells followed by pro-drug treatment were recently tested both in vitro and in vivo. Alternatively, the introduction of a gene that could specifically induce the EBV lytic cycle or adenovirus-delivered ribozymes to block EBV-induced B cell proliferation were also tested. These approaches, however, suffer from several limitations: 1) the effect is local and could only be exerted on the cells expressing the suicide gene or the cells nearby, 2) they often have a pronounced bystander effect of toxicity, and 3) they can only be used as therapeutic, but not as prophylactic methods.

None of these limitations apply to vaccination-based strategies, and the interest in vaccination against EBV has recently been increasing (1, 7, 12, 16, 17, 18, 19, 20, 21, 22).

DNA vaccines represent the latest development in vaccination strategies (23). Direct introduction of pDNA into the cells of a living host leads to the generation of both humoral and cellular immune responses (24, 25, 26, 27, 28, 29, 30). Protective immunity in various animal models and for a number of pathogens or tumors has been shown to be elicited by immunization with bacterial plasmids expressing a gene from the causative agent. Gene gun-mediated delivery of pDNA-coated gold beads into animal tissues in vivo provided the first demonstration of genetic immunization (31). Later, it was also described that i.m. immunization with pDNA encoding the nuclear protein of the influenza virus resulted in protection from the disease (32).

Several candidate Ags may be targeted for the development of a pDNA-based vaccine against EBV (19). These include the viral envelope protein genes and the genes expressed during the lytic or latent phase of the virus cycle. Nonetheless, to be able to design an infection-limiting and therapy-oriented vaccine, one should take into consideration the nature of EBV-associated diseases, in which the latency- or growth transformation-associated genes are the only expressed potential targets (20, 21). Accordingly, we have chosen one of these genes, EBNA-4, as a candidate for testing the pDNA strategy-based vaccination. We demonstrate that gene gun or i.m. immunization with an EBNA-4-expressing plasmid (E4) alone or in combination with plasmids expressing either murine GM-CSF (G) or murine IFN-{gamma} ({gamma}) leads to partial or full protection against the outgrowth of S6C-E4, an originally nonimmunogenic tumor transfected with EBNA-4. This protection was kept for a long period of time, as tumor challenge after 3 mo did not lead to any tumor outgrowth in the E4-immunized mice. Furthermore, E4-immunized mice that have rejected S6C-E4 were also protected from a subsequent rechallenge with a control S6C tumor lacking EBNA-4 expression. Our work suggests that a pDNA vaccine based on the use of a growth transformation-associated EBV gene can induce a long-term memory T cell-mediated immune response that protects the host from tumor outgrowth. This pDNA-based vaccination may prove to be particularly useful for the prevention and possibly treatment of EBV-related diseases, including infectious mononucleosis, XLPS, PTLD, and AIDS-related polyclonal lymphoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines

ACA (H-2f) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They were propagated and held in our specific pathogen-free environment in the MTC animal house at the Karolinska Institute (Stockholm, Sweden). All experiments performed have been approved by the animal ethical committee at the Karolinska Institute.

The S6C cell line was derived from a spontaneous mammary adenocarcinoma that has been originated in an ACA mouse (33). S6C-gpt and S6C-E4 are control plasmid and EBNA-4 transfectants, respectively (34). The MCB is a methylcholanthrene-induced fibrosarcoma of ACA origin. All cell lines were maintained by in vivo passage in syngenic ACA mice or in vitro in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM MEM, and 50 µM 2-ME, all from Life Technologies (Paisley, Scotland).

Plasmid construction and test

All genes were inserted in the pCDNA3 vector (Invitrogen BV, NV Leek, The Netherlands). The BamHI fragment including the full size EBNA-4 gene was excised from the pBabe (34) and cloned into the BamHI site in the multiple cloning sequence (MCS) of pCDNA3. The orientation was tested with restriction mapping, and the expression was confirmed with immunofluorescence staining after transfection by electroporation of the EBV-negative Burkitt lymphoma cell line BL-41 using a polyclonal human serum from a healthy donor.

An IFN-{gamma}-expressing plasmid (P3{gamma}) was constructed by excising the HindIII-BamHI fragment from the plasmid APtag-1 (kindly provided by Dr. T. Blankenstein, MDC, Berlin, Germany) and subcloning it into the corresponding site in the MCS of pCDNA3. A GM-CSF-expressing plasmid (P3G) was constructed by excising the HindIII-XbaI fragment from the plasmid RJB-GM (35) (kindly provided by Dr. H. Ertl, The Wistar Institute, Philadelphia, PA) and subcloning it into the corresponding sites in the MCS of pCDNA3. The orientation and identity of the resulting cytokine plasmids were confirmed with restriction mapping and with a specific ELISA for IFN-{gamma} (data not shown).

Immunization and challenge

Genetic immunization was accomplished by either gene gun or i.m. immunization. Plasmids were prepared from LB ampicillin Escherichia coli cultures using Qiagen plasmid giga kit (Qiagen GmbH, Hilden, Germany). Concentrations and purity were determined by spectrophotometry and with analytical gel electrophoresis. Mice were injected in the regenerating tibialis-anterior muscle, as described by Davis et al. (26), using a total of 40 µg pDNA/100 µl PBS/muscle of either control plasmid (P3), EBNA-4 expression plasmid plus control plasmid P3 (E4), EBNA-4 plus GM-CSF expression plasmids (E4G), or EBNA-4 plus IFN-{gamma} expression plasmids (E4{gamma}). Plasmids were mixed in equal molar quantities. Gene gun immunization was done using Accell helium-driven gene gun, as described earlier (27) using 2 µg DNA CaCl2 precipitated onto 1-µm gold beads (Bio-Rad, Richmond, CA) per shot and giving two nonoverlapping shots per mouse.

Both gene gun and i.m. immunized mice were boosted 2 mo after the initial immunization, followed by a second boosting 1 mo later.

Mice were alternatively immunized with irradiated (100 Gy) 106 tumor cells s.c. on three occasions with 1–2-wk intervals. Immunized mice were challenged 2 wk or 3 mo after the last boosting by s.c. injection of 5 x 103-104 tumor cells, as indicated in the figure legend. Tumors were measured by caliper, and the tumor sizes were scored using the formula: mean tumor diameter = diameter 1 (d1) + d2 + ... dn/n, in which n is the number of measured diameters. Mice were monitored for a period of at least 4 mo after challenge.

Proliferation test

Splenocytes were harvested from immunized mice. A single cell suspension was prepared and cells were resuspended in IMDM supplemented with 10% FBS, L-glutamine, and antibiotics. Mixed splenocytes and tumor cell cultures were prepared by mixing 3 x 106 splenocytes with 3 x 105 tumor cells/ml. Cultures were incubated for 5 days at 37°C in 7.5% CO2. Tritium-labeled thymidine (1 µCi) was added to each well of U-shaped bottom 96-well plates. Cells were further incubated for 18 h in the same conditions as above and harvested. The amount of incorporated tritium-labeled thymidine was measured using ß plate reader (Wallac, Turku, Finland). Test samples were set up in triplicates and the stimulation index (SI) was calculated using the formula: SI = experimental count in cultures stimulated with the EBNA-4 transfectant/experimental count in cultures stimulated with the gpt transfectant. SDs were <=10%.

Cytokine test

Mixed splenocyte-tumor cell cultures were prepared as for the proliferation test and incubated at 37°C in 7.5% CO2. Supernatants were collected after 3 days. Supernatants were tested for the presence of IFN-{gamma} and IL-4 using commercially available matched Ab pairs for mouse cytokine ELISA (ImmunoKontact, Bioggio, Switzerland, and BioSource, Fleurus, Belgium, respectively), according to the manufacturers’ procedures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasmid construction and the induction of specific antitumor immunity

To study the possibility of inducing an anti-EBV immune response using genetic immunization, we have constructed a human CMV immediate-early promoter-driven EBNA-4 expression vector (E4). This vector expresses the full size gene, and its product could be detected by indirect immunofluorescence using a polyclonal human serum from a healthy donor in transiently transfected EBV-negative BL 41 cells (data not shown). Cytokine-expressing plasmids were constructed using the same vector as that used for the EBNA-4. The S6C is a spontaneous nonimmunogenic murine mammary carcinoma of ACA (H-2f) origin. S6C transfected with EBNA-4, EBNA-5, latent membrane protein 2A (LMP2A), and LMP2B can induce rejection reactions in syngeneic mice when immunized with the relevant irradiated tumor transfectant (34). Mice immunized with E4 either by i.m. injection or by gene gun were partially protected from challenge with S6C-E4 and E4-transfected S6C cells (Figs. 1God and 2b). While all control (P3) immunized mice had to be sacrificed because of progressively growing tumors, 43% of the i.m. and 50% of the gene gun E4- immunized mice did not develop any palpable tumor (Fig. 1Go, a–d and Fig. 2Go, a and b). To investigate the possibility of improving this protection, we have coinjected or bombarded the mice with a mixture of E4 and the GM-CSF expression plasmid (E4G) or IFN-{gamma} expression plasmid (E4{gamma}). An increase in the protection was observed in the mice receiving i.m. immunization with E4G or E4{gamma} as compared with those immunized with E4 only, as 67% of the mice in these groups were protected from the tumor (Fig. 1Go, e and f). The inclusion of the cytokine-expressing plasmids in the immunization did not lead to a nonspecific rejection of the tumor, as mice immunized with the P3 plasmid in combination with these plasmids (P3G and P3{gamma}) could not reject the tumor (Fig. 1Go, b and c).



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  		FIGURE 1. Intramuscular immunizations with E4, E4G, or E4{gamma} protect from tumor outgrowth. Mice were immunized with 40 µg of P3 (15 mice), P3G (7 mice), or P3{gamma} (7 mice) plasmids (a–c) as controls or with E4 (14 mice), E4G (6 mice), or E4{gamma} (6 mice) plasmids (d–f) three times, as described in Materials and Methods. Two weeks after the last immunization, mice were challenged with 104 S6C-E4 tumor cells s.c. Each curve represents the tumor growth in an individual mouse. The percentage of the mice that have rejected the tumor is indicated at the x-axis.

 


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  		FIGURE 2. Gene gun immunization protects from tumor outgrowth. Mice (8 per group) were immunized with P3 (a) as a control or with E4, E4G, or E4{gamma} (b–d) plasmids three times, as described in Materials and Methods. Two weeks after the last immunization, mice were challenged with 104 S6C-E4 tumor cells s.c. Each curve represents the tumor growth in an individual mouse. The percentage of the mice that have rejected the tumor is indicated at the x-axis.

 
Mice immunized by gene gun with E4G or E4{gamma} combinations were more protected as compared with those immunized with the same plasmids by i.m. immunizations or with E4 by gene gun, as most of the mice in the E4G (88%) and all of the E4{gamma}-immunized mice were protected (Fig. 2Go, c and d). Interestingly, in both gene gun and i.m. immunized mice, the tumor growth latency period was prolonged in the E4, E4G, and E4{gamma} immunized as compared with the control P3, P3G, and P3{gamma} in the i.m. immunized mice (Fig. 1Go, a–f), and as compared with the control P3 in the gene gun immunized mice (Fig. 2Go, a–d). Thus, the tumor did not start to grow in these mice until it had reached 20 mm of diameter in most of the mice in the control groups, and not until 2–5 wk later did the tumors reach a mean diameter of 20 mm in the E4-, E4G-, and E4{gamma}-immunized mice (Figs. 1Go, a–f and 2, a–d). All of the protected mice remained tumor free for up to 6 mo after the challenge (data not shown).

Genetic immunization leads to a long-term protective memory response

To study whether this immunization approach would lead to a long-term memory response associated with the protection, we immunized the mice by gene gun with P3 or E4{gamma}. Three months after, the last immunization mice were challenged with S6C-E4. Mice immunized with P3 were not protected from the tumor outgrowth as the tumor grew and reached a large volume in all of them (Fig. 3Goa). Interestingly, mice immunized with E4{gamma}, however, were fully protected and no tumor has grown in any of these mice (Fig. 3Gob). This provides for the first time an evidence for a suggested long-term memory associated with pDNA immunization, which leads to the protection of a tumor outgrowth. Similar data were obtained using i.m. immunization (data not shown).



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  		FIGURE 3. Gene gun immunization results in a long-term memory, which protects from tumor outgrowth. Mice were immunized with a, P3 (6 mice) as a control, or with b, E4{gamma} (13 mice) plasmids three times with 1–2-mo intervals. Three months after the last immunization, mice were challenged with 5 x 103 S6C-E4 tumor cells s.c. Each curve represents the tumor growth in an individual mouse. The percentage of the mice that have rejected the tumor is indicated at the x-axis.

 
The development of cross-protective immunity

To investigate the possibility of inducing antiparental S6C tumor immunity in the mice that have rejected S6C-E4 as a result of E4 pDNA immunization, 10 mice of those that had survived the S6C-E4 challenge were rechallenged with 104 of the control transfectant S6C-gpt tumor cells. Tumor development was delayed in the rechallenged mice, and up to 50% of them were fully protected from the development of this nonimmunogenic tumor (Fig. 4Goa). All protected mice remained tumor free for more than 6 mo after the rechallenge (data not shown). As a control, we used mice immunized three times with irradiated S6C-E4. All of these mice immunized with irradiated tumor cells developed large tumors and were sacrificed within 5–9 wk from the challenge date (Fig. 4Goa). This cross-protection was not due to the development of a nonspecific immune response induced by pDNA immunization, since a subsequent challenge of the mice that have rejected S6C-E4 with the unrelated chemically induced tumor MCB resulted in the development of large tumors, and those mice were then sacrificed within 5–7 wk after challenge, similar to that in naive mice (Fig. 4Gob).



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  		FIGURE 4. Mice that have rejected the S6C-E4 tumor have developed cross-protective immunity specific against the parental tumor S6C-gpt. a, Ten of the mice that have rejected the S6C-E4 tumor (Fig. 2Go, b–d) were rechallenged with 104 S6C-gpt tumor cells s.c. on the other flank (empty squares). A group of six mice that have been immunized three times with 106 of the irradiated S6C-E4 tumor cells was used as a control (filled diamonds). b, Seven of the mice that have rejected the S6C-E4 tumor (Fig. 3Gob) were challenged with 104 MCB tumor cells s.c. on the other flank (empty squares). A group of four naive mice was used as a control (filled diamonds). Mice were sacrificed when the tumor size reached a 20 mm diameter. Percent survival refers to the percentage of mice that were still alive at the corresponding time point.

 
The induction of EBNA-4-specific T cell-mediated immune response

To study the immune response induced by E4 immunization, we tested the proliferative and cytokine responses by T cells from the immunized mice. We detected a specific T cell proliferative response as a result of stimulating spleen cells from the E4-, E4G-, and E4{gamma}-immunized mice with the S6C-E4 tumor in vitro, a response that paralleled the in vivo protection in magnitude. Splenocytes from E4-, E4G-, and E4{gamma}-immunized mice were preferentially stimulated by S6C-E4, yielding a proliferation index of 2.5–4 times as compared with that seen in cultures stimulated with S6C-gpt tumor cells (Fig. 5Go). This response was higher in the cultures of cells from mice coinjected with a cytokine expressing plasmids in addition to E4, and it was maximal (SI = 4) in the case of IFN-{gamma} codelivery (Fig. 5Go). There was no significant proliferation in the splenocyte cultures from control P3-immunized mice in response to S6C-E4 above that in response to S6C-gpt. Similar data were obtained using splenocytes from either i.m. or gene gun immunized mice.



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  		FIGURE 5. Splenocytes from E4, E4G, or E4{gamma} i.m. immunized mice proliferate specifically in response to S6C-E4 stimulation. Splenocytes from pDNA-immunized mice were cultured in the presence of either S6C-E4 or S6C-gpt control tumor cells. SI was calculated as described in Materials and Methods.

 
We then tested whether the E4 pDNA immunization would result in a Th1 or a Th2 memory response. To achieve this, the cytokines produced by splenocytes spontaneously or upon stimulation with S6C-E4 or S6C-gpt were compared. As illustrated in Table IGo, this response was strictly of the Th1 cell response, as indicated by the production of IFN-{gamma} upon stimulation with S6C-E4. No production of the Th2 cytokine IL-4 was detected. Here again, T cells from mice coinjected with E4 and IFN-{gamma} were the best specific responders in concurrence with the in vivo protection data (Table IGo). Similar data were obtained using splenocytes from either i.m. or gene gun immunized mice (data not shown).


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  		Table I. pDNA immunization results in a specific Th1 cytokine responsea

 
To test the possibility of inducing CTL responses to EBNA-4 by pDNA immunization, spleens or lymph node cells from EBNA-4 pDNA-immunized mice were stimulated with irradiated S6C-E4 or S6C-gpt tumor cells. Five days later, we used these cultures as effector cells in a cytotoxicity 51Cr release assay using S6C-E4 or S6C-gpt tumor cells as targets. We were unable to measure any CTL response in this test (data not shown). We did consider that the level of the EBNA-4 expression could have limited our CTL response detection. Accordingly, we also overexpressed EBNA-4 in S6C-E4 using vaccinia infection both when the tumor cells were used as stimulator and as target cells. We still were unable to measure any CTL response (data not shown). This could be due to either an inherent resistance of our tumor target cells to lysis by CTLs, or the immunization not inducing EBNA-4-specific CTL responses. In either case, this would argue that protection of the pDNA-immunized mice from outgrowth of the EBNA-4-expressing tumor was not associated with a CTL response.

Similarly, we were unable to detect a specific Ab response to EBNA-4 using an immunofluorescence technique with sera from pDNA-immunized mice as a potential source of EBNA-4-specific Abs and EBNA-4-positive cells as target for staining. Our inability to detect EBNA-4-specific Abs in the pDNA-immunized mice appears to be a specific case for this Ag, as we were able to induce a high titer Ab response with pDNA immunization using another Ag (the mycobacterial heat-shock 65; data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the search for tumor vaccines, those for cancers associated with viruses are predicted to be the first to become applied. The first licensed anti-cancer vaccine was developed by Hilleman, targeting the Marek’s disease virus from the same family as EBV, and resulting in a 100% effective protection from tumor growth in chicken (17). The development of a vaccine against EBV has started in the beginning of the last decade with the use of gp350 (1) (reviewed by Morgan (19)). This approach has proven to be partially successful in a variety of animal models, using vaccinia virus-based vaccines or recombinant proteins.

We present herein a novel approach for vaccination against EBV, based on the recently developed technique of DNA immunization and on the EBNA-4 Ag, an approach that offers several advantages. Our study shows that as a result of specific pDNA immunization, it is possible to achieve up to 100% protection from a tumor expressing the EBNA-4 gene. This is in contrast to the only partial protection observed when immunizing with whole irradiated S6C-E4 cells (34). The superior protection seen with pDNA immunization as compared with when immunizing with whole cells may depend on a prolonged antigenic exposure and/or a more efficient antigenic processing and presentation via the professional APC such as the dendritic cells (36). Remarkably, full protection was obtained even after 3 mo from the last immunization. This provides for the first time evidence for suggested long-term memory associated with pDNA immunization that leads to the protection of tumor outgrowth. This finding supports the applying of this method to vaccinate at an early age against diseases, which could be encountered later in life.

Using cytokine-expressing plasmids, we were able to further enhance pDNA-based immunization, resulting in higher in vivo protection that correlated well with an increased in vitro T cell response. We can confirm the reported capacity of GM-CSF to potentiate pDNA immunization, probably due to enhancing the initiation of the immune response by recruiting professional APC to the site of the Ag expression, as suggested (35, 36, 37, 38, 39). Full protection against tumor outgrowth was only obtained by coimmunizing the mice with EBNA-4- and IFN-{gamma}-expressing plasmids. This marked effect of IFN-{gamma} in enhancing pDNA-induced protection from tumor outgrowth, as shown in this study for the first time, may depend on the induction of a strong Th1 response. Studies by others showed no effect of this cytokine in pDNA-induced antiviral immune responses (35). This could be explained by a Th2-induced Ab-mediated antiviral response that could not be enhanced by IFN-{gamma}, or as shown recently by its effect on the promoter driving the expression of the studied Ag (37). We have used a CMV promoter to drive the expression of EBNA-4 in this study, and this promoter in contrast with MHC promoters has not been described to be affected by IFN-{gamma} (37).

It is noteworthy that the gene gun approach has resulted in a better protection than what is seen with i.m. immunization. The i.m. immunization is mediated by bone marrow-derived dendritic cells or macrophages as APC (40, 41). These APC are either directly transfected with the injected plasmid or they uptake the Ag from the transfected myocytes. Gene gun immunization results in the in vivo transfection of the skin-derived dendritic cells, which later localize to lymph nodes (42, 43). The enhanced protection provided by gene gun immunization could therefore be due to increased availability of the Ag at the site of the primary immune response, the lymph nodes. In line with this suggestion, Boyle et al. have recently provided evidence that targeting the Ag encoded by DNA vaccine to the lymph nodes resulted in an enhanced immune response (44). Alternatively, this difference could be explained by the higher variation in the i.m. immunization as compared with gene gun immunization. This variation was suggested to be due to the high likelihood of misses of inoculations or due to the 10–100 times lesser expression of the gene when using i.m. as compared with gene gun immunizations (45).

The protection provided by pDNA immunization with EBNA-4-expressing plasmid was specific and was not due to a nonspecific effect of pDNA since control pDNA-immunized mice were not protected (Figs. 1Go, a–f and 2, a–d).

Lukacs et al. have previously demonstrated the induction of cross-protective antitumor immune response as a result of introducing a strongly immunogenic (the mycobacterial heat-shock protein 65 gene) transgene into originally nonimmunogenic tumor J-774 (46). They have shown that mice that have been immunized with this transgenic tumor became resistant to a subsequent challenge with the parental tumor, an effect that was not due to the development of nonspecific immunity, since tumors other than the parental one were not rejected by the transgenic-tumor immunized mice. We herein confirmed that finding by using a different system, which might be of significant interest for vaccine development regarding the possible emergence of EBNA-4-negative variants of tumor cells that would be resistant to immune response directed against EBNA-4. Our data suggest that such a variant could be detected and eliminated, probably via an immune response that has developed during the process of the elimination of the EBNA-4-expressing tumor cells. It has also been reported recently that nonimmunogenic tumor cells pulsed with immunogenic peptide could prime mice to the rejection of this tumor even when the challenge is done by nonpulsed tumor cells (47). Therefore, the introduction of an immunogenic component into a nonimmunogenic tumor, which results in inducing a protective immune response against that tumor, represents an interesting tumor vaccination approach that deserves a further consideration.

We have not detected any Th2 cytokine production in either i.m. or gene gun immunization. This is in contrary to previously published data, in which gene gun immunization was shown to drive Th2 production as compared with Th1 production that was induced by i.m. immunization (48). This might be explained by the nature of our Ag as compared with influenza Ags, the amount of DNA used in our study that was 4 times higher, or simply due to a difference in the immune response between the different strain of mice used in these studies and the ACA strain that we used in our study (45, 48, 49).

Another advantage with the pDNA-based vaccination is the lack of immunogenicity of the vaccine construct (25, 50). This has been a considerable problem with vaccines based on viral vectors. The previous use of a vaccine against EBV based on vaccinia virus vector proved to have limited efficacy in a clinical study due to the presence of preexisting immunity to vaccinia in man (16). Also, in terms of safety, pDNA immunization is superior to virally based immunization methods (25, 50).

The pDNA approach would be particularly valid to test in EBV-negative patients with the need to receive solid organ transplants from EBV-positive donors, a patient group at high risk to develop EBV-related PTLD (51). In this context, it is worthwhile to mention that it has been shown that immunotherapy is very successful in treating and preventing EBV-associated PTLD (52). Remarkably, pDNA immunization was shown to break the tolerance normally associated with neonate immunization (53). This is particularly important as a significant number of the PTLD occur in pediatric liver transplantation, in which the majority of the recipients are <15 mo old and EBV seronegative (20). It is also appropriate to suggest testing this noninfectious pDNA vaccine in XLPS patients. The recent identification of the XLP gene would allow the diagnosis for individuals at risk of XLPD before their exposure to EBV, and vaccinating them (54). These patients are mainly infected at an early age, resulting in death of up to 85% by 10 years of age and full mortality by the age of 40. EBNA-4 is the safest Ag to use in a pDNA vaccine-based approach when choosing among the EBV-latent proteins (55, 56). It is the only one that has not been associated with transformation abilities, and we did not notice any apparent abnormality in the E4-immunized mice as compared with control P3 or nonimmunized mice (our unpublished data).

Taken together, the present study provides a rational basis for testing the efficacy of an EBNA-4-based pDNA vaccine that could provide protection against those EBV-associated diseases in which EBNA-4 is expressed. This study should also stimulate efforts to evaluate other EBV gene-based pDNA vaccines. One such vaccine could be based on the gp350-encoding gene (19), which might be able to prevent EBV infection via the production of neutralizing Abs.


   Acknowledgments
 
We thank Mrs. Margareta Hagelin, Maj-Lis Solberg, Mia Löwbeer, Lena Norenius, and Endre Kobold for excellent technical assistance.


   Footnotes
 
1 This work was supported by grants from the Swedish Cancer Society, the Cancer Society of Stockholm, and the King Gustaf V Jubilee Fund. J.C. is a recipient of a student fellowship from the Karolinska Institute, and A-M.T.C. is a recipient of a student fellowship from the Swedish Cancer Society. Back

2 Address correspondence and reprint requests to Dr. Jehad Charo, Microbiology and Tumorbiology Centre (MTC), Karolinska Institute, Box 280, S-17177, Stockholm, Sweden. E-mail address: Back

3 Current address: Department of General Pathology, University of Rome, Italy. Back

4 Abbreviations used in this paper: XLPS, X-linked lymphoproliferative syndrome; BL, Burkitt’s lymphoma; EBNA, EBV nuclear Ag; MCS, multiple cloning sequence; pDNA, plasmid DNA; PTLD, posttransplant lymphoproliferative disorder; SI, stimulation index. Back

Received for publication April 13, 1999. Accepted for publication September 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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