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[Cancer Research 64, 400-405, January 1, 2004]
© 2004 American Association for Cancer Research

Immunology

Enhancement of DNA Vaccine Potency by Coadministration of a Tumor Antigen Gene and DNA Encoding Serine Protease Inhibitor-6

Tae Woo Kim1, Chien-Fu Hung1, David A. K. Boyd1, Liangmei He1, Cheng-Tao Lin1, Dion Kaiserman5, Phillip I. Bird5 and T-C. Wu1234

1Departments of Pathology, 2Oncology, 3Obstetrics and Gynecology, and 4Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, and 5Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Serine protease inhibitor 6 (SPI-6), also called Serpinb9, inhibits granzyme B and thus may provide a method for delaying apoptotic cell death in dendritic cells. We have previously enhanced DNA vaccine potency by targeting antigen to MHC antigen presentation pathways, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, domain II of Pseudomonas aeruginosa exotoxin A, or the sorting signal of the lysosome-associated membrane protein type 1. In this study, we explored intradermal coadministration of DNA encoding SPI-6 with DNA constructs encoding human papillomavirus type 16 E7 linked to these intracellular targeting molecules for its ability to generate E7-specific CD8+ T-cell immune responses and E7-specific antitumor effects. This combination of strategies resulted in significantly increased E7-specific CD8+ T-cell and CD4+ Th1-cell responses, enhanced tumor treatment ability, and stronger tumor protection when compared with vaccination without SPI-6. Among these targeting strategies tested, mice vaccinated with Sig/E7/lysosome-associated membrane protein type 1 mixed with SPI-6 showed the greatest fold increase in E7-specific CD8+ T cells (~5-fold). Vaccination with a nonfunctional mutant of SPI-6 did not result in immune enhancement, indicating that enhancement was dependent on the antiapoptotic function of SPI-6. Our results suggest that DNA vaccines combining strategies that enhance MHC class I and II antigen processing with SPI-6 have potential clinical implications for control of viral infection and neoplasia.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA vaccines provide a means of administering antigen to the immune system and initiating cell-mediated immune responses. Intradermal administration of DNA vaccines via gene gun has proven to be the most effective delivery technique because it allows for the direct delivery of DNA encoding antigen of interest into dendritic cells (DCs; Ref. 1 ), the most potent professional antigen-presenting cells. DCs process the antigen and travel to draining lymph nodes where antigenic epitopes are presented to naïve CD8+ and CD4+ T cells, stimulating them to develop into CTLs and T-helper cells, respectively (2) . The ability of the gene gun to directly target DCs with DNA vaccine constructs has allowed us to evaluate strategies for improving DNA vaccine potency by enhancing antigen processing and presentation by DCs.

We have previously enhanced MHC class I and MHC class II antigen processing by DCs via several intracellular targeting strategies designed to route human papillomavirus type 16 (HPV-16) E7 antigen to desired subcellular compartments. These strategies include linking DNA encoding E7 antigen to DNA encoding Mycobacterium tuberculosis heat shock protein 70 (3) , calreticulin (4) , domain II of Pseudomonas aeruginosa exotin A (5) , or the sorting signal of the lysosome-associated membrane protein 1 (LAMP-1; Ref. 6 ). We have previously shown that mice vaccinated with any of these strategies linked to E7 display enhanced E7-specific CD8+ T-cell responses and antitumor effects when compared with mice vaccinated with wild-type E7 DNA alone.

We have recently explored the use of antiapoptotic proteins to enhance DNA vaccine potency by prolonging DC life (7) . DCs have a limited life span that hinders their long-term ability to prime antigen-specific T cells (8) . A principal contributor to the short life of DCs is CTL-induced apoptosis. CTLs are programmed to recognize antigens and kill the cells expressing them. Because DCs express MHC I:antigen peptide complexes, newly primed CTLs sometimes kill the very DCs that activated them (9) . Interrupting CTL-induced apoptosis and thereby prolonging the survival of DCs may facilitate the priming of antigen-specific CD8+ T cells and increase cell-mediated immune responses.

To this end, we investigated the capacity of the serine protease inhibitor (SPI-6; Ref. 10 ) to delay CTL-induced DC death. CTLs can secrete granzyme B (GrB) and perforin (11) , which act in concert as part of granule-mediated apoptosis, the dominant pathway of CTL-induced apoptosis (for review, see Ref. 12 ). The serine protease inhibitors (serpins) represent a potential solution to the problem of CTL suicide (13 , 14) and CTL-induced DC apoptosis by inactivating GrB (for reviews of serpin classification and regulation, see Refs. 15 , 16 ). Medema et al. (9) demonstrated that maturing DCs naturally express SPI-6 to inhibit GrB-triggered apoptosis and showed that overexpression of SPI-6 provides DCs with a powerful mechanism for defense against cytotoxic T cells.

On the basis of these findings, we hypothesized that coadministration of an SPI-6 expression vector with various DNA vaccines would prolong DC life, enhancing antigen presentation by DCs and, therefore, the immune response. Our data indicated that coadministering DNA encoding SPI-6 with DNA encoding intracellular targeting molecules linked to E7 prolongs DC life by delaying apoptosis of DCs and enhances cell-mediated E7-specific immune responses and antitumor effects to a greater extent than either of these strategies alone. These results suggest that a vaccination strategy combining intracellular targeting with SPI-6 to prolong DC life may additionally enhance DNA vaccine potency and may have important future clinical applications.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid DNA Constructs and DNA Preparation.
The generation of pcDNA3-E7 (3) , pcDNA3-calreticulin /E7 (4) , pcDNA3- domain II of Pseudomonas aeruginosa exotoxin A/E7 (5) , pcDNA3-E7/heat shock protein 70 (3) , and pcDNA3-Sig/E7/LAMP-1 (6) has been described previously. For generation of pcDNA3-SPI-6, SPI-6 was first amplified with PCR using mouse cDNA as the template and a set of primers, 5'-cccgaattcatgaatactctgtctgaagga-3' and 5'-ttggatcctggagatgagaacctgccaca-3'. The amplified product was then cloned into the EcoRI/BamHI sites of the pcDNA3 vector.

To generate the inactive mutant SPI-6 (mtSPI-6) containing the P14 mutation (T327R), most of the SPI-6 open reading frame was amplified from pSVTf/SPI-6 (10) using the primers 5'-ggctgctgcagcctcccggccttcctcattgat-3' (antisense) and 5'-gcatcatgaatactctgtc-3' (sense) and cloned into pZeroblunt (Invitrogen). The product included a naturally occurring PstI site downstream of the primer-introduced T327R substitution. This partial open reading frame was cloned into the EcoRI site of pSVTf, and the full-length open reading frame was then reconstituted by inserting a 200-bp PstI fragment containing the last part of the open reading frame and 3' untranslated region and verified by DNA sequencing. For generation of pcDNA3-mtSPI-6, mutant SPI-6 was cut at the EcoRI/BamHI sites from pSVTf-mtSPI-6 and cloned into the EcoRI/BamHI sites of the pcDNA3 vector. The accuracy of these constructs was confirmed by DNA sequencing. The DNA was amplified in Escherichia coli DH5{alpha} and purified as described previously (3) . The expression of SPI-6 and mtSPI-6 in COS-7 cells transfected with DNA encoding antiapoptotic protein was characterized by reverse transcription-PCR.

Mice.
Six to 8-week-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and kept 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 (3) . 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 p.s.i. C57BL/6 mice (5/group) were immunized with 2 µg of pcDNA3-encoding E7, calreticulin/E7, E7/heat shock protein 70, domain II of Pseudomonas aeruginosa exotoxin A/E7, or Sig/E7/LAMP-1 and mixed with 2 µg of pcDNA3, pcDNA3-SPI-6, or pcDNA3-mtSPI-6. The mice received a booster with the same dose 1 week later.

Intracellular Cytokine Staining and Flow Cytometry Analysis.
Cell surface marker staining of CD8 or CD4 and intracellular cytokine staining for IFN-{gamma} or interleukin (IL)-4, as well as flow cytometry analysis were performed under conditions described previously (3) . Splenocytes were harvested from mice 1 week after the last vaccination. Before intracellular cytokine staining, 4 x 106 pooled splenocytes from each vaccination group were incubated for 16 h with either 1 µg/ml E7 peptide (RAHYNIVTF) containing an MHC class I epitope for detecting E7-specific CD8+ T-cell precursors or 10 µg/ml E7 peptide (amino acid 30–67) containing an MHC class II epitope for detecting E7-specific CD4+ T-cell precursors. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA).

In Vivo Tumor Protection and Tumor Treatment Experiments.
The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously (17) . For the tumor protection experiments, C57BL/6 mice (5/group) were s.c. challenged with 5 x 104 TC-1 tumor cells/mouse in the right leg 1 week after the last vaccination. Mice were monitored for evidence of tumor growth by palpation and inspection twice a week.

To study the subsets of lymphocytes that are important for the antitumor effects, a tumor protection experiment was performed, coupled with in vivo antibody depletion using a protocol similar to one previously described (4) . Briefly, mice (5/group) were vaccinated, boosted 1 week later, and challenged with 5 x 104 TC-1 tumor cells 2 weeks after boosting. Antibody depletion was initiated 1 week before tumor challenge and continued until sacrifice. Monoclonal antibody GK1.5 was used for CD4 depletion. Monoclonal antibody 2.43 was used for CD8 depletion. Monoclonal antibody PK136 was used for natural killer depletion. Mice were monitored twice a week and sacrificed on day 42 after tumor challenge.

For the tumor treatment experiment, mice (5/group) were challenged with 1 x 105 TC-1 tumor cells/mouse in the tail vein to simulate hematogenous spread of tumors (18) . Mice were treated with DNA 3 days after tumor challenge. Mice were monitored twice a week and sacrificed on day 42 after the last vaccination. The mean number of pulmonary nodules in each mouse was evaluated by experimenters blinded to sample identity.

Survival of DC Line (DC-1).
The immortalized DC line was kindly provided by Dr. Kenneth Rock (University of Massachusetts, Worcester, MA; Ref. 19 ). With continued passage, we have generated subclones of DCs (DC-1) that are easily transfected using Lipofectamine 2000 (Life Technologies, Inc., Rockville, MD). DC-1 (5 x 105) was cotransfected with 1 µg of pcDNA3-E7/GFP mixed with 4 µg of pcDNA3-SPI-6, mt SPI-6, or no insert after the formation of Lipofectamine 2000/DNA complexes. GFP+ cells were collected 16 h after cotransfection using fluorescence-activated cell sorting. Twenty thousand GFP+ DC-1 cells were incubated with 2 x 106 cells of the E7-specific CD8+ T-cell line for 6 h. To determine the percentage of apoptotic DCs, Annexin V staining was performed after gating around a population of GFP+ cells and analyzed via flow cytometry analysis as described above.

Data Analysis.
All data expressed as means ± SD are representative of at least two different experiments. Data for tumor treatment experiments were evaluated by ANOVA. Comparisons between individual data points were made using a 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 use of the method of Kaplan and Meier and by use of the log-rank statistic. All Ps <0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Coadministering DNA Encoding E7 with DNA Encoding SPI-6 Increases CD8+ T-Cell Responses and Enhances Antitumor Effects.
To test our hypothesis that SPI-6 prolongs DC life and increases the immune response elicited by vaccination with E7-encoding DNA constructs, we coadministered pcDNA3-E7 with pcDNA3 or pcDNA3-SPI-6. Fig. 1ACitation shows that vaccination with pcDNA3-E7 coadministered with pcDNA3-SPI-6 generated a greater number of E7-specific IFN-{gamma}-secreting CD8+ T cells (32.3 ± 5.1/3 x 105 splenocytes) than coadministration with pcDNA3 (7.0 ± 1.0/3 x 105 splenocytes) or vaccination with pcDNA3-E7 alone (10.7 ± 1.5/3 x 105 splenocytes). These data indicate that SPI-6 DNA is capable of enhancing E7-specific CD8+ T-cell responses when coadministered with E7 DNA.



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  		Fig. 1. Determination of E7-specific CD8+ T-cell precursors generated and tumor protection elicited in mice vaccinated with pcDNA3-E7 mixed with psDNA3-SPI-6 or pcDNA3. Mice were immunized with pcDNA3-E7 coadministered with pcDNA3-serine protease inhibitor 6 or pcDNA3 and received a booster with the same dose 1 week later. Splenocytes were harvested 1 week after the last vaccination. pcDNA3 with no insert was used as a negative control. A, bar graph depicting the number of E7-specific IFN-{gamma}-secreting CD8+ T-cell precursors/3 x 105 splenocytes (mean ± SD). B, in vivo tumor protection experiment. Mice were challenged with 5 x 104 TC-1 tumor cells 1 week after the last vaccination. C, in vivo antibody depletion experiments to determine the contribution of various lymphocyte subsets to the tumor protection generated by administration of pcDNA3-E7 mixed with pcDNA3-serine protease inhibitor 6. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge.

 
To determine whether the observed enhancement in E7-specific CD8+ T-cell-mediated immunity elicited by coadministration of SPI-6 DNA with E7 DNA leads to significant E7-specific antitumor effects, we performed an in vivo tumor protection experiment using a previously described E7-expressing tumor model, TC-1 (17) . As shown in Fig. 1BCitation , 60% of mice vaccinated with pcDNA3-E7 coadministered with pcDNA3-SPI-6 remained tumor-free 42 days after TC-1 challenge. All of the mice vaccinated with pcDNA3-E7 mixed with pcDNA3, however, developed tumors after only 14 days. The percentage of tumor-free mice vaccinated with E7 DNA mixed with SPI-6 DNA was found to be significantly higher than the percentage of tumor-free mice vaccinated with E7 DNA and empty vector (P < 0.014). These findings suggest that coadministration of E7 DNA with SPI-6 DNA can lead to enhanced antitumor effects against an E7-expressing tumor cell line.

To determine the subsets of lymphocytes that are important for the observed antitumor effect generated by vaccination with E7 DNA and SPI-6 DNA, we performed an in vivo antibody depletion experiment (Fig. 1C)Citation ; all of the mice depleted of CD8+ T cells grew tumors within 2 weeks of TC-1 challenge. In contrast, 40% of the mice with CD4+ or natural killer depletion and 60% of the mice with no depletion remained tumor-free 42 days after TC-1 challenge. These data indicate that CD8+ T cells play a vital effector role in the antitumor defense generated by the DNA vaccine. The percentage of tumor-free mice in the CD8-depleted group was significantly lower than the percentage of tumor-free mice in the nondepleted group (P < 0.013). CD4+ and natural killer cells may also contribute to the antitumor effect, although the numbers of tumor-free CD4 and NK-depleted mice are not significantly different from the number of tumor-free nondepleted mice.

Combining Intracellular Targeting Strategies with SPI-6 DNA Significantly Enhances E7-Specific CD8+ T-Cell-Mediated Immune Responses.
Given its proposed mechanism of immune enhancement, coadministration of SPI-6 can likely enhance other DNA vaccination strategies. Therefore, we hypothesized that coadministering DNA encoding E7 and intracellular targeting strategies with DNA encoding SPI-6 would additionally enhance E7-specific CD8+ T-cell immune responses. We coadministered SPI-6 with E7 linked to domain II of Pseudomonas aeruginosa exotoxin A, heat shock protein 70, calreticulin, or the sorting signal of LAMP-1. As depicted in Fig. 2, A and BCitation , the enhanced immune response elicited by DNA vaccines encoding intracellular targeting strategies is additionally improved when the vaccines are coadministered with DNA encoding SPI-6. Each of the constructs generated a greater number of CD8+ T cells when coadministered with SPI-6 DNA than when coadministered with empty vector. The vaccine encoding Sig/E7/LAMP-1 produced the greatest SPI-6-related enhancement (~5-fold) of the E7-specific CD8+ T-cell precursors generated. These data suggest that coadministering DNA encoding intracellular targeting strategies with DNA encoding SPI-6 can enhance the E7-specific CD8+ T-cell response in vaccinated mice and that coadministering Sig/E7/LAMP-1 DNA with SPI-6 DNA results in the greatest fold enhancement of this immune response.



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  		Fig. 2. Determination of IFN-{gamma}-secreting, E7-specific CD8+ T-cell precursors in mice vaccinated with DNA encoding E7 linked to intracellular targeting molecules mixed with pcDNA3 or pcDNA3- serine protease inhibitor 6 (SPI-6). Mice were immunized with pcDNA3 (control), pcDNA3-E7, pcDNA3-Sig/E7/lysosome-associated membrane protein 1 (LAMP-1), pcDNA3-domain II of Pseudomonas aeruginosa exotoxin A [ETA(dII)]/E7, pcDNA3-E7/heat shock protein 70 (HSP70), or pcDNA3-calreticulin (CRT)/E7 coadministered with pcDNA3 or with pcDNA3-SPI-6, and splenocytes were collected 1 week after the last vaccination. A, representative figure of the flow cytometry data. The data presented in this figure are from one representative experiment of two performed. B, bar graph depicting the number of antigen-specific IFN-{gamma}-secreting CD8+ T-cell precursors/3 x 105 splenocytes (mean ± SD). The data from mice vaccinated with pcDNA3 and pcDNA3-E7 is the same as the data presented in Fig. 1ACitation .

 
Coadministering SPI-6 with Various Intracellular Targeting Strategies Significantly Enhances CD4+ Th 1 Cell-Mediated Immune Responses but not CD4+ Th2 Cell-Mediated Immune Responses.
To assess the ability of SPI-6 to enhance the generation of E7-specific CD4+ T cells, we performed intracellular cytokine staining for IFN-{gamma} and IL-4 with flow cytometry analysis. As depicted in Fig. 3ACitation , coadministering DNA encoding SPI-6 with DNA encoding E7 linked to intracellular targeting molecules increased the E7-specific CD4+ Th1 cell response elicited. Coadministering DNA encoding SPI-6 with DNA encoding Sig/E7/LAMP-1 generated the highest frequency of E7-specific IFN-{gamma}-secreting CD4+ Th1 cell precursors (77.0 ± 3.6/3 x 105 splenocytes), displaying a significant increase (by ~5-fold) over the response elicited by Sig/E7/LAMP-1 coadministered with empty vector (14.1 ± 1.0/3 x 105 splenocytes).



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  		Fig. 3. Characterization of Th1 and Th2 E7-specific CD4+ T-cell precursors in mice vaccinated with DNA encoding E7 linked to intracellular targeting molecules mixed with pcDNA3 or pcDNA3-serine protease inhibitor 6 (SPI-6). Mice were immunized with pcDNA3, pcDNA3-E7, pcDNA3-Sig/E7/lysosome-associated membrane protein 1 (LAMP-1), pcDNA3-domain II of Pseudomonas aeruginosa exotoxin A [ETA(dII)]/E7, pcDNA3-E7/heat shock protein 70 (HSP70), or pcDNA3-calreticulin (CRT)/E7 coadministered with pcDNA3 or with pcDNA3-SPI-6. Splenocytes from vaccinated mice were harvested 7 days after a booster vaccination, cultured in vitro with MHC class II-restricted E7 peptide (amino acid 30–67) overnight, and stained for CD4, IFN-{gamma}, and interleukin (IL)-4. A, bar graph depicting the number of E7-specific IFN-{gamma}-secreting CD4+ T-cell precursors/3 x 105 splenocytes (mean ± SD). B, bar graph depicting the number of E7-specific IL-4-secreting CD4+ T lymphocytes/3 x 105 splenocytes (mean ± SD) in mice vaccinated as above. MICK-2 was used as a positive control (data not shown).

 
As shown in Fig. 3BCitation , coadministering the various E7 constructs with SPI-6 DNA did not increase the E7-specific CD4+ Th2 immune response. The number of E7-specific IL-4-secreting CD4+ T-cell precursors elicited by the vaccines decreased slightly when the E7 constructs were coadministered with SPI-6 DNA. These results suggest that SPI-6 is ineffective for enhancing Th2 CD4+ cell responses. Taken together, our findings indicate that vaccination with E7 DNA coadministered with SPI-6 DNA facilitates the activation of E7-specific IFN-{gamma}+ CD4+ Th1 cells but does not significantly affect the activation of E7-specific IL-4+ CD4+ Th2 cells.

Coadministering pcDNA3-Sig/E7/LAMP-1 with pcDNA3-SPI-6 Elicits Potent Antitumor Treatment Effects.
We sought to combine the intracellular targeting benefits of Sig/E7/LAMP-1 (6) with the proposed antiapoptotic benefits of SPI-6 to further test our hypothesis that SPI-6 DNA enhances antitumor effects and cell-mediated immune responses against E7 antigen. We chose Sig/E7/LAMP-1 over the other adjuvant constructs because vaccination with Sig/E7/LAMP-1 DNA showed the greatest fold increase in E7-specific CD8+ T cells and CD4+ Th1 cells when coadministered with SPI-6 DNA (Figs. 2BCitation and 3A)Citation .

To test the therapeutic effects of coadministration of Sig/E7/LAMP-1 DNA and SPI-6 DNA, we performed an in vivo tumor treatment experiment using a hematogenous spread pulmonary tumor model (18) . As shown in Fig. 4Citation , mice immunized with Sig/E7/LAMP-1 DNA coadministered with SPI-6 DNA exhibited significantly fewer pulmonary tumor nodules (3.6 ± 5.3) compared with naïve mice (118.6 ± 15.0; P < 0.001). Furthermore, coadministering Sig/E7/LAMP-1 DNA with SPI-6 DNA resulted in significantly fewer tumor nodules than coadministering Sig/E7/LAMP-1 DNA with empty vector (35.8 ± 12.9; P < 0.013). These results indicate that vaccination with Sig/E7/LAMP-1 DNA coadministered with SPI-6 DNA provides potent therapeutic effects against E7-expressing TC-1 tumor cells and that this tumor treatment is more effective than treatment provided by vaccination with Sig/E7/LAMP-1 DNA mixed with control backbone DNA.



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  		Fig. 4. In vivo Tumor treatment experiment in mice vaccinated with pcDNA3-Sig/E7/lysosome-associated membrane protein 1 (LAMP-1) coadministered with pcDNA3 or pcDNA3-serine protease inhibitor 6 (SPI-6). In vivo tumor treatment experiments were performed using the hematogenous spread lung model. Mice were challenged with 1 x 105 TC-1 cells/mouse and subsequently treated with pc-DNA3-Sig/E7/LAMP-1 coadministered with pcDNA3-SPI-6 or pcDNA3 3 days after tumor challenge. pcDNA3 mixed with pcDNA3-SPI-6 was used as a negative control. Data are expressed as the mean number of lung nodules ± SE.

 
The Antiapoptotic Function of SPI-6 Is Vital for Enhancing Cell-Mediated Immune Responses and Prolonging the Life of DCs.
The antiapoptotic function of a serpin can be destroyed by substituting the conserved P14 Thr with Arg (13) . To confirm that the antiapoptotic function of SPI-6 is required to prolong DC survival, we generated an inactive P14 mtSPI-6 and studied the effects of coadministration of Sig/E7/LAMP-1 DNA with mtSPI-6 DNA on the E7-specific CD8+ T-cell response in vaccinated mice. As shown in Fig. 5ACitation , vaccination with pcDNA3-Sig/E7/LAMP-1 coadministered with mtSPI-6 DNA yielded fewer E7-specific CD8+ T-cell precursors (132.0 ± 2.6/3 x 105 splenocytes) than vaccination with pcDNA3-Sig/E7/LAMP-1 coadministered with pcDNA3-SPI-6 (620.7 ± 22.9/3 x 105 splenocytes). Our results indicate that the antiapoptotic function absent in mtSPI-6 is critical for the observed enhancement of E7-specific CD8+ T-cell immune responses.



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  		Fig. 5. Determination of E7-specific CD8+ T-cell precursors in vivo and nonapoptotic dendritic cells (DCs) in vitro after coadministration of DNA encoding E7 with DNA encoding serine protease inhibitor 6 (SPI-6) or mutant SPI-6 (mtSPI-6). A, mice were immunized with pcDNA3-Sig/E7/lysosome-associated membrane protein 1 (LAMP-1) mixed with pcDNA3-SPI-6, pcDNA3-mtSPI-6, or pcDNA3. Mice were vaccinated, and splenocytes were prepared as described in Fig. 1Citation . Bar graph depicting the number of antigen-specific IFN-{gamma}-secreting CD8+ T-cell precursors/3 x 105 splenocytes (mean ± SD). B, DCs were transfected in vitro with pcDNA3-E7/GFP mixed with pcDNA3-SPI-6, pcDNA3-mtSPI-6, or pcDNA3. Annexin V staining and flow cytometry analysis were performed after gating around a GFP+ cell population. DCs were cocultured with an E7-specific CD8+ T-cell line. Bar graph depicting the percentage of Annexin V- GFP+ DCs detected. The data shown in this figure are from one representative experiment of two performed and are presented as the mean ± SD.

 
To confirm that SPI-6 is indeed antiapoptotic, we incubated a DC line (DC-1), transfected with E7/GFP DNA together with SPI-6 DNA, empty vector, or mtSPI-6 DNA, with an E7-specific CD8+ T-cell line in vitro. GFP+ DC-1 cells were subsequently stained with Annexin V to determine the percentage of apoptotic cells. DC-1 cells staining positively for Annexin V represented apoptotic cells. As shown in Fig. 5BCitation , the percentage of GFP+, Annexin V-negative DC-1 target cells was greater in the DC line transfected with E7/GFP DNA mixed with SPI-6 DNA (13.63 ± 0.97) than in the DC line transfected with E7/GFP DNA mixed with empty vector or mtSPI-6 DNA. These data indicate that there were more nonapoptotic cells present in the DC-1 line transfected with SPI-6 DNA than in the DC-1 line transfected with mtSPI-6 DNA. Cotransfection with mtSPI-6 yielded nearly the same percentage of Annexin V-negative DC-1 cells as the empty vector (6.10 ± 0.30 and 6.67 ± 1.29, respectively), suggesting that mtSPI-6 is ineffective for prolonging DC survival.

We have previously shown that CD11-enriched cells from the draining lymph nodes of mice vaccinated with E7/GFP DNA coadministered with DNA encoding BCL-xL had more viable GFP+ CD11+ cells and compared with mice vaccinated with E7/GFP DNA coadministered with mtBCL-xL DNA, which has been minimally mutated to abolish its apoptotic function (7) . In the current study, we have performed similar experiments. We found that CD11+ cells from the draining lymph nodes of mice vaccinated with E7/GFP DNA and SPI-6 DNA have more viable cells and can activate more E7-specific CD8+ T cells than CD11+ cells from mice vaccinated with E7 DNA and mtSPI-6 DNA (data not shown). Taken together, our data suggest that SPI-6 does possess an antiapoptotic function and that this ability to delay apoptosis helps to enhance the E7-specific immune response elicited by DCs in vivo and prolong the life of DCs in vitro.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that coadministering DNA encoding E7 and intracellular targeting strategies with DNA encoding SPI-6 significantly enhances the potency of HPV-16 E7-encoding DNA vaccines and that the antiapoptotic function inherent in SPI-6 is vital to this enhancement. This DNA vaccine strategy elicited strong E7-specific CD8+ and IFN-{gamma}-secreting CD4+ T-cell responses and also generated significant antitumor effects. These results indicate that coadministering E7 DNA with SPI-6 DNA may help to control E7-expressing tumors and HPV infection.

DNA vaccines encoding E7 linked to intracellular targeting strategies and coadministered with SPI-6 DNA significantly enhanced the number of CD8+ T-cell precursors generated (Figs. 1Citation and 2)Citation . The most likely explanation for this increase is the role SPI-6 plays in preventing CTL-induced apoptosis in DCs. We used an inactive mtSPI-6 to confirm the importance of the antiapoptotic function of SPI-6 in the generation of antigen-specific CD8+ T cells. The SPI-6 mutant has a substitution in its proximal hinge, which destroys its ability to inhibit GrB and prevent GrB-mediated apoptosis. Thus, prolonged DC life as a result of inhibition of apoptosis by SPI-6 is responsible for the observed increase in the E7-specific CD8+ T-cell response.

In this study, we have observed increased numbers of E7-specific CD4+ Th1 cells (Fig. 2)Citation . CD4+ Th1 cells may contribute to the observed antitumor effect. Th1 cells are capable of stimulating the maturation of DCs via IFN-{gamma} secretion and CD40/CD40 ligand interaction (20) . Maturation of DCs by Th1 cells causes DCs to express IL-12 and prime antigen-specific CD8+ T cells more effectively. IL-12 secretion has been shown to significantly contribute to antitumor effects in vivo (21) . Thus, Th1 CD4+ T cells may augment the antitumor effect by stimulating DCs to produce IL-12, by secretion of IFN-{gamma} and by enhancing CTL activation by DCs.

We have previously transfected DCs with DNA encoding antiapoptotic proteins other than SPI-6 (including Bcl-xL and Bcl-2; Ref. 7 ). Vaccination with DNA encoding E7 mixed with DNA encoding these antiapoptotic proteins proved to be a powerful tool for enhancing the E7-specific CD8+ T-cell response and strengthening immune memory in vaccinated mice. We have shown that this enhancement was because of prolonged DC survival, resulting in enhanced antigen presentation to T cells by DCs in the draining lymph nodes. Antiapoptotic proteins of the Bcl-2 family (Bcl-2 and Bcl-xL) were found to contribute to the greatest enhancement of the E7-specific cell-mediated immune response. The use of these antiapoptotic proteins, however, raises serious safety concerns. Proteins of the Bcl-2 family are known to be overexpressed in some cancers and therefore have been implicated as contributors to cellular immortalization (22) .

In an effort to resolve these safety issues, we investigated the capacity of SPI-6 to prevent CTL-induced DC death by inhibiting the perforin/GrB mechanism of CTL-induced apoptosis. Because it is naturally expressed in mature DCs, SPI-6 may represent a potentially safe and effective method for enhancing DNA vaccine potency by offering a means of prolonging DC life without risk of DC immortalization (9) . Although the Bcl-2 antiapoptotic proteins inhibit CTL-induced apoptosis via multiple pathways (23 , 24) , SPI-6 and its human counterpart, PI-9, inhibit only the perforin/GrB pathway (10 , 13) . The other major pathway, Fas-mediated apoptosis, is not affected by SPI-6 (25) . In this way, SPI-6 may represent a means for inhibiting CTL-induced apoptosis without completely depriving CTLs of their capacity to trigger death in DCs. However, the safety profile of SPI-6 has yet to be explored as a DNA vaccine in a clinical context. There may be additional clinical safety concerns associated with SPI-6 that have not yet come to light.

Although SPI-6 may alleviate the safety concerns associated with Bcl-2 family proteins, Bcl-2 family proteins such as Bcl-xL provide a greater enhancement of DNA vaccine potency (26) . This is likely because Bcl-2 and Bcl-xL inhibit apoptosis at multiple points, whereas SPI-6 interferes solely with GrB activity. It is now clear that the granzyme family is composed of members other than GrB. This raises the possibility of enhancing DNA vaccine potency by coadministration of DNA encoding multiple granzyme inhibitor molecules with DNA encoding target antigen. Because perforin is important for the apoptotic function of the granzyme family, it may be possible to further inhibit apoptosis by disrupting perforin function. Therefore, by focusing on the perforin/granzyme pathway, it may be possible to design future DNA vaccines capable of safely inhibiting apoptosis to an extent equal to or greater than the inhibition by Bcl-2 or Bcl-xL.

In summary, the role of SPI-6 as a naturally expressed antiapoptotic factor offers an effective and potentially safer approach for prolonging the life of DCs and thus represents a means of increasing the potency of DNA vaccines. Our findings indicate that coadministering E7 DNA with SPI-6 DNA prolongs DC life and enhances E7-specific CD8+ T-cell activity and elicits strong antitumor effects against an E7-expressing tumor cell line in vivo. In addition, we have shown that a DNA vaccine strategy combining intracellular targeting with SPI-6 can successfully elicit strong E7-specific CTL and antitumor responses in a murine model. Because a majority of cervical cancers express HPV-16 E7, coadministration of E7 DNA vaccines with SPI-6 DNA may have useful clinical applications, offering a means of safely preventing and treating HPV infection and HPV-associated cervical lesions.


   ACKNOWLEDGMENTS
 
We thank Jessica Yeatermeyer for her assistance in preparation of the manuscript. We also thank Drs. Robert J. Kurman, Drew M. Pardoll, and Keerti Shah for helpful discussions and Dr. Richard Roden for critical review of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Grant support: National Cancer Institute, the Cancer Research Institute, the American Cancer Society (to T-C. W.), and the National Health and Medical Research Council, Australia (to P. I. B.).

Note: Drs. Kim and Hung contributed equally to this work.

Requests for reprints: Dr. T-C. Wu, Department of Pathology, The Johns Hopkins University School of Medicine, Richard Ross Research Building, Room 512H, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 614-3899; Fax: (410) 287-4295; E-mail: wutc{at}jhmi.edu

Received 5/23/03. Revised 8/28/03. Accepted 10/16/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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