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The Journal of Immunology, 2003, 171: 2970-2976.
Copyright © 2003 by The American Association of Immunologists

Enhancing DNA Vaccine Potency by Combining a Strategy to Prolong Dendritic Cell Life with Intracellular Targeting Strategies 1

Tae Woo Kim2,*, Chien-Fu Hung2,*, David Boyd*, Jeremy Juang*, Liangmei He*, Jeong Won Kim*, J. Marie Hardwick{dagger},{ddagger},§,¶ and T.-C. Wu3,*,{dagger},¶,||

Departments of * Pathology, {dagger} Molecular Microbiology and Immunology, {ddagger} Pharmacology and Molecular Sciences, § Neurology, Oncology, and || Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD 21205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-xL, prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-xL with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8+ T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-xL DNA showed the greatest increase in E7-specific CD8+ T cells (~13-fold increase). This combination of strategies resulted in increased CD8+ T cell functional avidity, an increased E7-specific CD4+ Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-xL DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Deoxyribonucleic acid vaccines have become a promising approach to Ag-specific immunotherapy due to their ease of preparation, stability, and capacity for repeated administration without significant generation of vector-specific humoral immunity (1). Intradermal administration of DNA vaccines via gene gun has been shown to be the most effective means of DNA vaccine delivery, because it allows professional APCs to be directly transfected with the vaccine construct (2). Professional APCs, such as dendritic cells (DCs), 4 are a vital factor in cell-mediated immunity, because they are capable of activating naive CD8+ and CD4+ T lymphocytes in the draining lymph nodes, stimulating them to develop into CTLs and Th cells, respectively (3).

Using a gene gun administration system, we have developed DNA vaccines using intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded human papillomavirus (HPV)16 E7 Ag by DCs to T cells (for review, see Ref. 4). These strategies have been developed to compensate for the weak immune response generated by DCs transfected with DNA encoding only wild-type E7 Ag. Some of these strategies include linking E7 Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) (5), to calreticulin (CRT) (6), or to the sorting signal of the lysosome-associated membrane protein 1 (LAMP-1) (7). We have previously shown that mice vaccinated with any of these strategies display enhanced E7-specific CD8+ T cell responses and antitumor effects when compared with mice vaccinated with wild-type E7 DNA alone.

As another means of modifying DCs to enhance the potency of naked DNA vaccines, we have previously transfected DCs with DNA encoding antiapoptotic proteins (Bcl-xL, Bcl-2, XIAP, dn caspase-8, and dominant-negative caspase-9) (8). Vaccination with DNA encoding E7 with DNA encoding antiapoptotic proteins has proven to be a powerful tool for enhancing the E7-specific CD8+ T cell response and strengthening immune memory in vaccinated mice. This enhancement was shown to be due to prolonged DC survival, resulting in enhanced Ag presentation to T cells by DCs in the draining lymph nodes. Of these previously examined antiapoptotic proteins, DNA encoding Bcl-xL was found to be responsible for the strongest enhancement of the immune response. We have shown that the antiapoptotic function of Bcl-xL appears to be important, because mutant (mt)Bcl-xL, which has lost its antiapoptotic function (9), loses the ability to prolong DC life and enhance DNA vaccine potency.

Because intracellular targeting and antiapoptotic strategies modify DCs via different mechanisms, we proposed that it might be feasible to combine antiapoptotic strategies for prolonging DC life with intracellular targeting strategies for enhancing MHC class I and II presentation of Ag in DCs. The success of this strategy could provide us with a powerful tool for treating and preventing HPV infection and HPV-associated lesions. To explore this possibility, we have examined the effects on the observed murine E7-specific immune response of coadministration of DNA encoding Bcl-xL with various DNA constructs incorporating different intracellular targeting strategies with E7. We have found that these combinations enhance the E7-specific immune response in vaccinated mice to a greater degree than either antiapoptotic or intracellular targeting strategies alone. This combination strategy also results in greater enhancement of the CD8+ immune response in CD4 knockout (CD4KO) mice than vaccination with intracellular targeting strategies alone in wild-type mice. These results suggest that a vaccination strategy combining intracellular targeting with a strategy to prolong DC life may further enhance DNA vaccine potency and may also have significant future clinical implications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid DNA constructs and DNA preparation

The generation of pcDNA3, pcDNA3-E7, pcDNA3-Sig/E7/LAMP-1 (7), pcDNA3-CRT/E7 (6), and pcDNA3-HSP70/E7 (5) has been described previously. pSG5 plasmids encoding Bcl-xL or mt 7 (our mtBcl-xL) were generated as described previously (9). The DNA was amplified and purified as described previously (5).

Mice

Six- to 8-wk-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, Maryland). Six- to 8-wk old C57BL/6 CD4KO female mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, MD). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.

DNA vaccination

DNA-coated gold particles were prepared according to a previously described protocol (5). DNA-coated gold particles were delivered to the shaved abdominal region of mice using a helium-driven gene gun (Bio-Rad, Hercules, CA) with a discharge pressure of 400 psi. C57BL/6 mice were immunized with 2 µg of pcDNA3 encoding E7, CRT/E7, E7/HSP70, or Sig/E7/LAMP-1 mixed with 2 µg of pSG5 or pSG5-Bcl-xL. The mice received a booster with the same dose 1 wk later.

Intracellular cytokine staining and flow cytometry analysis

Splenocytes were harvested from mice (five per group) 1 or 7 wk (for memory T cells) after the last vaccination. Before intracellular cytokine staining, 4 x 106 pooled splenocytes from each vaccination group were incubated overnight with 1 µg/ml E7 (RAHYNIVTF) peptide containing an MHC class I epitope (aa 49–57) for detecting E7-specific CD8+ T cell precursors or 1 µg/ml E7 peptide containing an MHC class II epitope (aa 30–67) for detecting E7-specific CD4+ T cell precursors. Intracellular IL-4 and IFN-{gamma} staining and flow cytometry analysis were performed as described previously (5). Analysis was performed on a FACScan with CellQuest software (BD Biosciences, Mountain View, CA).

For the determination of the avidity of E7-specific CD8+ T cells, mice were vaccinated with pcDNA3-Sig/E7/LAMP-1 with pSG5-no insert, with pSG5-Bcl-xL, or with pSG5-mtBcl-xL. Mice were boosted with the same vaccine 1 wk after the first vaccination. Splenocytes were collected and pooled 1 wk after the booster. The pooled splenocytes were incubated with different concentrations of E7 peptide (aa 49–57; 1, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, or 10-8 µg/ml) overnight. The number of E7-specific IFN-{gamma}-secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis as described above.

In vivo tumor treatment and long-term tumor protection

The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously (10). In brief, HPV-16 E6, E7, and ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate TC-1. The mean number of pulmonary nodules in each mouse was evaluated by experimenters blinded to sample identity.

For the tumor treatment experiments, mice (five per group) were challenged with 1 x 105 TC-1 tumor cells/mouse in the tail vein to simulate hematogenous spread of tumors (7). Mice were treated with DNA 3 days after tumor challenge. Mice were monitored twice per week and sacrificed on day 42 after tumor challenge.

To study the subsets of lymphocytes that are important for the antitumor effects, a tumor protection experiment was performed, coupled with in vivo Ab depletion using a protocol similar to one previously described (11). Briefly, mice were vaccinated, boosted 1 wk later, and challenged with 1 x 104 TC-1 tumor cells 1 wk after boosting. Ab depletion was initiated concurrently with tumor challenge and continued until sacrifice. mAb GK1.5 was used for CD4 depletion. mAb 2.43 was used for CD8 depletion. mAb PK136 was used for NK depletion. Mice were monitored twice per week and sacrificed on day 42 after tumor challenge.

For the long-term tumor protection experiments, mice (five per group) were i.v. challenged with 1 x 104 TC-1 tumor cells 7 wk after the last vaccination. Mice were monitored twice per week and sacrificed on day 42 after tumor challenge.

Statistical analysis

All data expressed as means ± SE are representative of at least two different experiments. Data for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by ANOVA. Comparisons between individual data points were made using Student’s t test. In the tumor protection experiment, the principal outcome of interest was time to development of tumor. The event time distributions for different mice were compared by Kaplan and Meier and by log-rank analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Combined antiapoptotic and intracellular targeting strategies can further enhance E7-specific CD8+ T cell responses in vaccinated mice

To explore whether DNA encoding Bcl-xL is capable of enhancing DNA vaccines using the various intracellular targeting strategies, we coadministered Bcl-xL with E7 linked to HSP70, CRT, or LAMP-1. As shown in Fig. 1, A and B, coadministration of Bcl-xL with any of the three intracellular targeting strategies resulted in an increase in IFN-{gamma}-secreting E7-specific CD8+ T cell precursors compared with coadministration with pSG5 empty vector. CRT/E7 mixed with Bcl-xL produced the strongest response, but Sig/E7/LAMP-1 mixed with Bcl-xL displayed the greatest fold increase in the number of IFN-{gamma}-secreting E7-specific CD8+ T cell precursors (at least a 10-fold increase). Our data indicate that coadministration with Bcl-xL can be used in combination with any of the intracellular targeting strategies to further enhance DNA vaccine potency, and that coadministration of Bcl-xL DNA with Sig/E7/LAMP-1 DNA displays the greatest fold increase of the E7-specific CD8+ T cell immune response.



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  		FIGURE 1. Intracellular cytokine staining followed by flow cytometry analysis to determine the E7-specific CD8+ T cell response in mice vaccinated with DNA using intracellular targeting strategies mixed with pSG5-Bcl-xL or pSG5. Mice were immunized with pcDNA3, pcDNA3-E7, pcDNA3-E7/HSP70, pcDNA3-Sig/E7/LAMP-1, or pcDNA3-CRT/E7 coadministered with pSG5 or with pSG5-Bcl-xL. Splenocytes from vaccinated mice were harvested 7 days after a booster vaccination, cultured in vitro with MHC class I-restricted E7 peptide (aa 49–57) overnight, and stained for both CD8 and IFN-{gamma}. A, Representative figure of the flow cytometry data. The data presented in this figure are from one experiment of two performed. B, Bar graph depicting the difference in the number of E7-specific IFN-{gamma}-secreting CD8+ T cells between coadministration with pSG5 and with pSG5-Bcl-xL. pcDNA3 empty vector mixed with pSG5 and pSG5-Bcl-xL was used as a negative control.

 
Coadministration of pcDNA3-Sig/E7/LAMP-1 with pSG5-Bcl-xL increases the avidity of the E7-specific CD8+ T lymphocyte immune response

Prior studies have shown that high-avidity CTL provide better protection against viral infection (12) and tumor challenge (13) than low-avidity CTL. In addition, duration of DC and T cell interaction has been implicated to be important for the generation of high-avidity T cells (14). Thus, we performed a functional avidity assay to determine the avidity of E7-specific CD8+ T cells generated by vaccination with Sig/E7/LAMP-1 mixed with Bcl-xL, mtBcl-xL, or empty vector. We defined the number of IFN-{gamma}-secreting CD8+ T cells stimulated by 1 µg/ml E7 peptide (aa 49–57) as a maximum response and compared the functional avidity of T cells from mice vaccinated with Bcl-xL or empty vector at 50% of the maximum. We found that the concentration of E7 peptide required to achieve 50% of the maximum IFN-{gamma}+CD8+ T cell response was ~4 x 10-5 µg/ml for mice vaccinated with Sig/E7/LAMP-1 with Bcl-xL, and ~3 x 10-3 µg/ml for mice vaccinated with Sig/E7/LAMP-1 mixed with empty vector or mtBcl-xL (Fig. 2). Therefore, coadministration of Sig/E7/LAMP-1 with Bcl-xL generates higher-avidity E7-specific CD8+ T cells than coadministration of Sig/E7/LAMP-1 with empty vector or mtBcl-xL in vaccinated mice. Furthermore, because the functional avidity of E7-specific CD8+ T cells elicited by vaccination with mtBcl-xL was nearly identical with that elicited by vaccination with empty vector, it is likely that the antiapoptotic function of Bcl-xL was responsible for the observed increase in functional avidity.



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  		FIGURE 2. Functional avidity of E7-specific CD8+ T cells in mice vaccinated with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5 or pSG5-Bcl-xL. Mice were immunized with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL, pSG5-mtBcl-xL, or pSG5. Splenocytes were collected 1 wk after vaccination and incubated with different concentrations of E7 peptide (aa 49–57) for 20 h. pcDNA3 mixed with pSG5 encoding Bcl-xL was used as a negative control. Data were acquired for comparison of concentrations of peptide needed to generate 50% of maximum CD8+ T cell response in mice vaccinated with DNA encoding Bcl-xL, mtBcl-xL, or empty vector.

 
Vaccination with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL generated an enhanced Th1 and a reduced Th2 E7-specific CD4+ T cell immune response

The ability of the LAMP-1 targeting strategy to enhance Ag presentation to CD4+ T lymphocytes is achieved through targeting of expressed Ag to endosomal/lysosomal compartments, important loci for the MHC class II Ag presentation pathway (15). To determine the nature of the E7-specific CD4+ T cell response to vaccination with Sig/E7/LAMP-1 mixed with Bcl-xL or empty vector, we performed intracellular cytokine staining for IFN-{gamma} (secreted by Th1 cells) or IL-4 (secreted by Th2 cells) with flow cytometry analysis using mouse splenocytes derived 1 wk after the last vaccination. As shown in Fig. 3, vaccination with Sig/E7/LAMP-1 mixed with Bcl-xL generated significantly more E7-specific Th1 CD4+ T lymphocytes (86.3 ± 14.3 vs 13.5 ± 2.5/3 x 105 splenocytes) and fewer E7-specific Th2 CD4+ lymphocytes (43.4 ± 3.8 vs 65.2 ± 6.4/3 x 105 splenocytes) than vaccination with Sig/E7/LAMP-1 mixed with empty vector. These data suggest that coadministration with DNA encoding Bcl-xL may contribute to an enhanced E7-specific CD4+ Th1 cell response and a reduced E7-specific CD4+ Th2 cell response.



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  		FIGURE 3. Intracellular cytokine staining followed by flow cytometry analysis to characterize Th1 and Th2 CD4+ T cell responses generated in mice vaccinated with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL or pSG5. Splenocytes from vaccinated mice were harvested 7 days after a booster vaccination, cultured in vitro with MHC class II-restricted E7 peptide (aa 30–67) overnight, and stained for CD4, IFN-{gamma}, and IL-4 as described in Materials and Methods. A, Bar graph depicting the number of E7-specific IFN-{gamma}-secreting CD4+ T cell precursors/3 x 105 splenocytes. B, Bar graph depicting the number of E7-specific IL-4-secreting CD4+ T cell precursors/3 x 105 splenocytes.

 
Vaccination with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL results in a stronger E7-specific CD8+ T cell response in CD4KO mice than vaccination with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5 in wild-type mice

To examine whether CD4+ T cells are essential for the observed enhancement of the CD8+ T cell immune response, we assessed the number of E7-specific CD8+ T cells generated in C57BL/6 and C57BL/6 CD4KO mice vaccinated with Sig/E7/LAMP-1 mixed with Bcl-xL or empty vector. As shown in Fig. 4, A and B, we observed that wild-type mice vaccinated with Sig/E7/LAMP-1 mixed with Bcl-xL induced a greater E7-specific CD8+ T cell response than in wild-type mice vaccinated with Sig/E7/LAMP-1 mixed with empty vector. The same trend was observed in the C57BL/6 CD4KO mice where CD4KO mice vaccinated with Sig/E7/LAMP-1 mixed with Bcl-xL generated more E7-specific CD8+ T cells than CD4KO mice vaccinated with Sig/E7/LAMP-1 mixed with empty vector. When we compare the CD4KO mice with the wild type mice, we see that vaccination with Sig/E7/LAMP-1 mixed with Bcl-xL resulted in a ~10-fold greater E7-specific CD8+ T cell response in wild-type C57BL/6 mice compared with the response to the same vaccine strategy in CD4KO mice. This suggests that CD4+ T cells contribute significantly to the observed E7-specific CTL response.



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  		FIGURE 4. E7-specific CD8+ T lymphocyte response in CD4KO mice vaccinated with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5 encoding Bcl-xL or no insert. C57BL/6 and C57BL/6 CD4KO mice were immunized with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL or pSG5. Splenocytes were collected and prepared as described in Fig. 1. The number of E7-specific IFN-{gamma}-secreting CD8+ T cell precursors was analyzed by intracellular cytokine staining followed by flow cytometry analysis. A, Flow cytometry data depicting numbers of E7-specific IFN-{gamma}-secreting CD8+ T cells in mice after vaccination. The data are from one experiment of two performed. B, Bar graph depicting the number of E7-specific IFN-{gamma}-secreting CD8+ T cell precursors/3 x 105 splenocytes.

 
Although there was a considerable decrease in the number of E7-specific CD8+ T cells generated in CD4KO mice compared with the number of E7-specific CD8+ T cells generated in wild-type mice vaccinated with the same regimen, our data demonstrated that the coadministration of Bcl-xL DNA with Sig/E7/LAMP-1 DNA in CD4KO mice was able to generate ~2-fold more E7-specific CD8+ T cells than coadministration of Sig/E7/LAMP-1 DNA with pSG5 in wild-type mice. These data suggest that a DNA vaccine combining an antiapoptotic strategy with an intracellular targeting strategy may be able to generate better CD8+ T cell-mediated immune responses in a CD4-depleted host than those generated by a DNA vaccine using only an intracellular targeting strategy in an immunocompetent host.

Coadministration of pcDNA3-Sig/E7/LAMP-1 with pSG5-Bcl-xL generates better antitumor treatment effects than coadministration of pcDNA3-Sig/E7/LAMP-1 with pSG5

A factor vital to the success of an HPV therapeutic vaccine is the ability to treat patients with HPV infection or HPV-related cancers. To determine the therapeutic effectiveness of our strategy, we tested the ability of Sig/E7/LAMP-1 mixed with Bcl-xL or empty vector to treat TC-1, an E7-expressing tumor cell line, using a previously described hematogenous spread model (7). As shown in Fig. 5A, mice treated with Sig/E7/LAMP-1 mixed with Bcl-xL developed a significantly lower number of tumor nodules than mice treated with Sig/E7/LAMP-1 mixed with empty vector, the control, or the naive mice. This indicates that coadministration with Bcl-xL generates better tumor treatment ability than coadministration with empty vector in a mouse model.



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  		FIGURE 5. In vivo tumor treatment experiments and in vivo Ab depletion experiments in mice vaccinated with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL or pSG5. A, In vivo tumor treatment experiments. Mice were challenged with 1 x 105 TC-1 tumor cells i.v. and immunized 3 days later. Bar graph depicts the number of pulmonary tumor nodules in mice after vaccination with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5 encoding Bcl-xL or no insert. pcDNA3 mixed with pSG5 was used as a control. B, In vivo tumor protection experiment using Ab depletion to determine the contribution of various lymphocyte subsets to tumor treatment generated by pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL. Data are expressed as mean number of pulmonary nodules; bars ± SE.

 
We also performed a tumor protection experiment with Ab depletion to determine the subset of T lymphocytes responsible for the antitumor response. Mice were vaccinated with Sig/E7/LAMP-1 mixed with Bcl-xL and were subsequently challenged with TC-1. We initiated depletion of subsets of lymphocytes concurrent with tumor challenge. In Fig. 5B, we see that mice depleted of CD8+ T cells display nearly the same degree of tumor growth as naive mice, and mice depleted of CD4+ T cells display slightly increased tumor growth compared with nondepleted mice. Mice depleted of NK cells did not generate a significantly different number of tumor nodules compared with mice with no depletion. These data suggest that CD8+ T cells are essential for the antitumor effect, and that CD4+ T cells may also contribute to the observed antitumor effect, although not as strongly as CD8+ T cells.

Immunization with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5-Bcl-xL generates significantly enhanced long-term immune response and antitumor protection in vaccinated mice

A successful vaccine must be capable of generating an effective long-term protective immune response. To assess the ability of our vaccination strategy to generate long-term E7-specific CD8+ T cell immune responses and protective antitumor effects against TC-1, we compared vaccination with Sig/E7/LAMP-1 mixed with Bcl-xL to vaccination with Sig/E7/LAMP-1 mixed with empty vector. Intracellular cytokine staining followed by flow cytometry analysis to determine E7-specific CD8+ T cells was performed 1 and 7 wk after immunization. As shown in Fig. 6A, Sig/E7/LAMP-1 mixed with Bcl-xL generated an ~7-fold higher E7-specific IFN-{gamma} CD8+ T lymphocyte response at 7 wk postimmunization than Sig/E7/LAMP-1 mixed with empty vector. Thus, coadministration of the Sig/E7/LAMP-1 vaccine construct with Bcl-xL generates a stronger long-term immune response than coadministration of Sig/E7/LAMP-1 with empty vector.



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  		FIGURE 6. E7-specific CD8+ memory T cell immune response and long-term tumor protection generated in mice vaccinated with Bcl-xL DNA or empty vector. Mice were immunized with pcDNA3-Sig/E7/LAMP-1 mixed with pSG5 either encoding Bcl-xL or no insert. pcDNA3 mixed with pSG5 was used as a control. A, Bar graph depicting number of E7-specific IFN-{gamma} CD8+ T lymphocytes/3 x 105 splenocytes 1 and 7 wk after immunization. B, Long-term tumor protection experiment depicting number of pulmonary nodules in vaccinated mice. Mice were challenged with 1 x 104 TC-1 tumor cells 7 wk after immunization. Data are expressed as mean number of pulmonary tumor nodules; bars ± SE.

 
To determine the long-term tumor protection ability of our vaccination strategy, we challenged vaccinated mice with 1 x 104 TC-1 tumor cells 7 wk after the final immunization. As shown in Fig. 6B, mice vaccinated with Sig/E7/LAMP-1 mixed with Bcl-xL exhibited no pulmonary tumor nodules, whereas mice vaccinated with Sig/E7/LAMP-1 mixed with empty vector exhibited 1.6 ± 2.3 tumor nodules 42 days after TC-1 challenge. Therefore, coadministration of Sig/E7/LAMP-1 with Bcl-xL completely prevented tumor nodule formation 7 wk after vaccination. Taken together, our data indicate that a DNA vaccine combining an intracellular targeting strategy with a strategy that prolongs DC life generates a strong long-term E7-specific CD8+ T cell immune response and excellent long-term protective antitumor effects.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results support our hypothesis that a DNA vaccination strategy combining intracellular targeting with a strategy to prolong DC life is capable of enhancing Ag-specific immune responses to a greater degree than a DNA vaccination strategy using intracellular targeting strategies alone. We found this combination strategy to be effective with three vaccine strategies using intracellular targeting: HSP70/E7 (5), CRT/E7 (6), and Sig/E7/LAMP-1 (7), resulting in strong E7-specific CD8+ T cell responses as well as long-term tumor protection in vaccinated mice. These results may be attributed to prolonged DC life via inhibition of apoptosis by Bcl-xL, resulting in an increased quantity of longer-lived DCs in the draining lymph nodes (8), as well as to enhanced processing of Ag as a result of expression of CRT, LAMP-1, or HSP70 linked to E7, resulting in increased MHC:peptide presentation to T lymphocytes (5, 6, 7). Thus, our data indicate that it is possible to modify DCs simultaneously via two different mechanisms to further enhance DNA vaccine potency.

Of the targeting strategies tested, we observed that Sig/E7/LAMP-1 displayed the greatest enhancement of the E7-specific CD8+ T cell immune response when coadministered with Bcl-xL DNA (Fig. 1B). A possible explanation for this may be related to an increase in CD4 Th cells. The Sig/E7/LAMP-1 construct is the only one that targets Ag to the MHC class II processing pathway (15), activating Ag-specific CD4+ T cells more effectively than other constructs. Furthermore, in our study using CD4KO mice, we observed a significantly lower number of E7-specific CD8+ T cells in CD4KO mice than in wild-type mice. Thus, CD4+ T cells are important for the observed enhancement of the E7-specific CD8+ T cell response to vaccination with Sig/E7/LAMP-1.

Although CD4KO mice exhibited an overall reduced CD8+ T cell immune response when compared with wild-type mice vaccinated with the same regimen (Fig. 4, A and B), the coadministration of Bcl-xL DNA with Sig/E7/LAMP-1 DNA can enhance the E7-specific CD8+ T cell immune response in CD4KO mice when compared with CD4KO mice vaccinated with Sig/E7/LAMP-1 DNA with empty vector. Moreover, we showed that the coadministration of Bcl-xL DNA with Sig/E7/LAMP-1 DNA in CD4KO mice is able to generate more E7-specific CD8+ T cells than coadministration of Sig/E7/LAMP-1 DNA with pSG5 in wild-type mice. These results suggest that a DNA vaccination strategy combining intracellular targeting with antiapoptotic proteins is capable of enhancing the E7-specific CD8+ T cell response in individuals with a significant reduction in CD4+ T cell count. This could be clinically relevant to the treatment of AIDS patients, who are often handicapped by depleted CD4+ T cell levels. This depletion is a likely cause of the increased severity of HPV infection and associated lesions in HIV-positive subjects (for review, see Refs. 16 and 17). Thus, this combination strategy might prove useful for the control of HPV infection and HPV-associated lesions in CD4-depleted human subjects.

Coadministration of DNA encoding Bcl-xL resulted in the generation of higher-avidity E7-specific CD8+ T cells (Fig. 2). The antiapoptotic function of Bcl-xL was likely essential for this effect, because coadministration of mtBcl-xL DNA elicited E7-specific CD8+ T cells with a functional avidity nearly identical with those elicited by coadministration with empty vector. The ability of Bcl-xL to extend DC life span might lead to prolonged DC and T cell interaction in draining lymph nodes, and the duration of DC and T cell interaction has been implicated to be important for generating high-avidity Ag-specific T cells (14). Thus, prolonged DC life due to coadministration with Bcl-xL DNA may contribute directly to increased E7-specific CD8+ T cell avidity.

Ag-specific CD8+ T cells with high avidity are known to result in better qualitative Ag-specific immune protection and antitumor effects in vaccinated mice than low-avidity CD8+ T cells (18). High-avidity CD8+ T cells enhance protection by recognizing low Ag densities and killing infected cells sooner than low-avidity CD8+ T cells (12). Prior studies have shown that higher-avidity CTLs may produce a stronger antitumor effect in vaccinated mice (13, 19). Selective expansion of high-avidity Ag-specific T cells and memory T cell precursors may in turn enhance antitumor immunity. Thus, higher CD8+ T cell avidity may contribute to the strong antitumor effect that we observed in mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-xL DNA.

As HPV vaccine research has moved into the clinical arena, it has become relevant to discuss the clinical implications of newly developed HPV vaccines in anticipation of potential future clinical application. Safety is a major consideration for the clinical application of any new vaccination strategy and is especially important in this case, because increased Bcl-xL expression could be considered potentially hazardous to human subjects. This is due to the concern that the antiapoptotic effects of Bcl-xL could interfere with the normal regulation of DC function, which could result in autoimmunity. However, no evidence of autoimmunity was observed in any of the mice used in the study, based on histology of immunized mice (data not shown).

Oncogenesis is another relevant concern, because Bcl-xL has been implicated in oncogenic transformation of healthy cells (20). One strategy to improve safety is to transfect DCs with DNA encoding factors that may indirectly enhance DC survival with a reduced concern for oncogenicity, such as TNF-related activation-induced cytokine (21), CD40 ligand (22), IL-12 (23), and IL-15 (24), and serine protease inhibitor 6 (25). Among these molecules, CD40 ligand (26), IL-12 (27), and IL-15 (28) have been tested to enhance DNA vaccine potency. It would be interesting to evaluate the role that apoptotic inhibition plays in enhancing the Ag-specific immune responses mediated by these molecules in DNA vaccines.

In summary, we have shown that coadministration of DNA encoding an antiapoptotic protein with DNA encoding intracellular targeting strategies can further enhance the potency of DNA vaccines against an HPV-16 E7-expressing tumor line. This vaccine strategy led to an increased CD8+ T cell response, higher CD8+ T cell functional avidity, stronger immune memory, and enhanced antitumor effects in vaccinated mice when compared with vaccines using only intracellular targeting strategies. In addition, a DNA vaccine combining an antiapoptotic strategy with an intracellular targeting strategy may be able to generate better CD8+ T cell-mediated immune responses in a CD4-depleted host than those generated by DNA vaccination using only the intracellular targeting strategy in an immunocompetent host. These results encourage the application of antiapoptotic proteins in combination with other vaccine enhancement strategies for future development of therapeutic DNA vaccines to combat HPV infection and cervical cancer in the clinical realm.


   Acknowledgments
 
We thank Drs. Robert J. Kurman, Drew M. Pardoll, and Keerti Shah for helpful discussions. We thank Dr. Richard Roden for critical review of the manuscript.


   Footnotes
 
1 This work was supported by the National Cancer Institute, Cancer Research Institutes, and the American Cancer Society. Back

2 T.W.K. and C.-F.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. T.-C. Wu, Department of Pathology, Johns Hopkins University School of Medicine, Ross 512H, 720 Rutland Avenue, Baltimore, MD 21205. E-mail address: wutc{at}jhmi.edu Back

4 Abbreviations used in this paper: DC, dendritic cell; HPV, human papillomavirus; HSP70, heat shock protein 70; CRT, calreticulin; LAMP-1, lysosome-associated membrane protein 1; CD4KO, CD4 knockout; mt, mutant. Back

Received for publication April 25, 2003. Accepted for publication July 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gurunathan, S., D. M. Klinman, R. A. Seder. 2000. DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 18:927.[Medline]
  2. Condon, C., S. C. Watkins, C. M. Celluzzi, K. Thompson, L. D. Falo, Jr. 1996. DNA-based immunization by in vivo transfection of dendritic cells. Nat. Med. 2:1122.[Medline]
  3. Cella, M., F. Sallusto, A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10.[Medline]
  4. Moniz, M., M. Ling, C. F. Hung, T. C. Wu. 2003. HPV DNA vaccines. Front. Biosci. 8:D55.
  5. Chen, C. H., T. L. Wang, C. F. Hung, Y. Yang, R. A. Young, D. M. Pardoll, T. C. Wu. 2000. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res. 60:1035.[Abstract/Free Full Text]
  6. Cheng, W. F., C. F. Hung, C. Y. Chai, K. F. Hsu, L. He, M. Ling, T. C. Wu. 2001. Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. J. Clin. Invest. 108:669.[Medline]
  7. Ji, H., T. L. Wang, C. H. Chen, S. I. Pai, C. F. Hung, K. Y. Lin, R. J. Kurman, D. M. Pardoll, T. C. Wu. 1999. Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine human papillomavirus type 16 E7-expressing tumors. Hum. Gene Ther. 10:2727.[Medline]
  8. Kim, T. W., C. F. Hung, M. Ling, J. Juang, L. He, J. M. Hardwick, S. Kumar, T. C. Wu. 2003. Enhancing DNA vaccine potency by co-administration of DNA encoding anti-apoptotic proteins. J. Clin. Invest. 112:109.[Medline]
  9. Cheng, E. H., B. Levine, L. H. Boise, C. B. Thompson, J. M. Hardwick. 1996. Bax-independent inhibition of apoptosis by Bcl-xL. Nature 379:554.[Medline]
  10. Lin, K. Y., F. G. Guarnieri, K. F. Staveley-O’Carroll, H. I. Levitsky, J. T. August, D. M. Pardoll, T. C. Wu. 1996. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 56:21.[Abstract/Free Full Text]
  11. Cheng, W. F., C. H. Hung, C. Y. Chai, K. F. Hsu, L. He, M. Ling, T. C. Wu. 2001. Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen. J. Virol. 75:2368.[Abstract/Free Full Text]
  12. Derby, M., M. Alexander-Miller, R. Tse, J. Berzofsky. 2001. High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL. J. Immunol. 166:1690.[Abstract/Free Full Text]
  13. Cheng, W. F., C. F. Hung, S. I. Pai, K. F. Hsu, L. He, M. Ling, T. C. Wu. 2002. Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects. J. Biomed. Sci. 9:675.[Medline]
  14. Langenkamp, A., G. Casorati, C. Garavaglia, P. Dellabona, A. Lanzavecchia, F. Sallusto. 2002. T cell priming by dendritic cells: thresholds for proliferation, differentiation and death and intraclonal functional diversification. Eur. J. Immunol. 32:2046.[Medline]
  15. Wu, T. C., F. G. Guarnieri, K. F. Staveley-O’Carroll, R. P. Viscidi, H. I. Levitsky, L. Hedrick, K. R. Cho, J. T. August, D. M. Pardoll. 1995. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc. Natl. Acad. Sci. USA 92:11671.[Abstract/Free Full Text]
  16. Kuhn, L., X. W. Sun, T. C. Wright, Jr. 1999. Human immunodeficiency virus infection and female lower genital tract malignancy. Curr. Opin. Obstet. Gynecol. 11:35.[Medline]
  17. Del Mistro, A., L. Chieco Bianchi. 2001. HPV-related neoplasias in HIV-infected individuals. Eur. J. Cancer 37:1227.
  18. Alexander-Miller, M. A., G. R. Leggatt, J. A. Berzofsky. 1996. Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. Proc. Natl. Acad. Sci. USA 93:4102.[Abstract/Free Full Text]
  19. Yang, S., G. P. Linette, S. Longerich, F. G. Haluska. 2002. Antimelanoma activity of CTL generated from peripheral blood mononuclear cells after stimulation with autologous dendritic cells pulsed with melanoma gp100 peptide G209-2M is correlated to TCR avidity. J. Immunol. 169:531.[Abstract/Free Full Text]
  20. Lebedeva, I., R. Rando, J. Ojwang, P. Cossum, C. A. Stein. 2000. Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity. Cancer Res. 60:6052.[Abstract/Free Full Text]
  21. Wong, B. R., R. Josien, S. Y. Lee, B. Sauter, H. L. Li, R. M. Steinman, Y. Choi. 1997. TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J. Exp. Med. 186:2075.[Abstract/Free Full Text]
  22. Esche, C., A. Gambotto, Y. Satoh, V. Gerein, P. D. Robbins, S. C. Watkins, M. T. Lotze, M. R. Shurin. 1999. CD154 inhibits tumor-induced apoptosis in dendritic cells and tumor growth. Eur. J. Immunol. 29:2148.[Medline]
  23. Ploemacher, R. E., P. L. van Soest, H. Voorwinden, A. Boudewijn. 1993. Interleukin-12 synergizes with interleukin-3 and steel factor to enhance recovery of murine hemopoietic stem cells in liquid culture. Leukemia 7:1381.[Medline]
  24. Bykovskaia, S. N., M. Buffo, H. Zhang, M. Bunker, M. L. Levitt, M. Agha, S. Marks, C. Evans, P. Ellis, M. R. Shurin, J. Shogan. 1999. The generation of human dendritic and NK cells from hemopoietic progenitors induced by interleukin-15. J. Leukocyte Biol. 66:659.[Abstract]
  25. Medema, J. P., D. H. Schuurhuis, D. Rea, J. van Tongeren, J. de Jong, S. A. Bres, S. Laban, R. E. Toes, M. Toebes, T. N. Schumacher, et al 2001. Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J. Exp. Med. 194:657.[Abstract/Free Full Text]
  26. Mendoza, R. B., M. J. Cantwell, T. J. Kipps. 1997. Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization. J. Immunol. 159:5777.[Abstract]
  27. Kim, J. J., V. Ayyavoo, M. L. Bagarazzi, M. A. Chattergoon, K. Dang, B. Wang, J. D. Boyer, D. B. Weiner. 1997. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen. J. Immunol. 158:816.[Abstract]
  28. Xin, K. Q., K. Hamajima, S. Sasaki, T. Tsuji, S. Watabe, E. Okada, K. Okuda. 1999. IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine. Vaccine 17:858.[Medline]

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