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[Cancer Research 62, 7234-7240, December 15, 2002]
© 2002 American Association for Cancer Research

Carcinogenesis

Both E7 and CpG-Oligodeoxynucleotide Are Required for Protective Immunity against Challenge with Human Papillomavirus 16 (E6/E7) Immortalized Tumor Cells

Involvement of CD4+ and CD8+ T Cells in Protection1

Tae-Yoon Kim, Han-Jeong Myoung, Ji-Hyun Kim, In-Sung Moon, Tai-Gyu Kim, Woong-Shick Ahn and Jeong-Im Sin2

Departments of Dermatology [T-Y. K.], Surgery [I-S. M.], Microbiology [T-G. K.], Obstetrics and Gynecology [W-S. A.], and Catholic Research Institutes of Medical Science [H-J. M., J-H. K., J-I. S.], The Catholic University of Korea, Seoul 137-040, Korea


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An important goal of immunotherapy against human papillomavirus (HPV) infection and the cervical cancer is to control viral infection and the cancer cell growth. Here we investigate the utility of HPV 16 E7 along with CpG-oligodeoxynucleotide (ODN) for protection against HPV-immortalized tumor cells using an animal model. E7+ODN coinjection showed a significant suppression of tumor growth at both prophylactic and therapeutic levels. However, no such effect was observed without addition of both E7 and ODN. We additionally evaluated levels of immune responses by E7+ODN coinjection. E7+ODN resulted in E7-specific antibody (IgG1, IgG2a, IgG2b, and IgG3) and T-helper cell proliferative responses significantly higher than E7 alone. However, CTL responses were induced only by E7+ODN. Moreover, IFN-{gamma} production was detected only in E7+ODN immunized groups in which IFN-{gamma} releasing CD4+ (T-helper 1 type) and CD8+ T cells (CTL) were induced only by E7+ODN. Moreover, tumor protection appears to be mediated by CD4+ and in most CD8+ T cells, as determined by in vivo T-cell subset depletion. Taken together, these data suggest that E7+ODN codelivery could be an effective approach to induce E7-specific protective immune responses as a possible immunotherapeutic strategy for cervical cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ODN3 containing unmethylated CpG motifs can activate B cells, monocytes, and natural killer cells, and induce a Th1 like pattern of cytokine production (1, 2, 3, 4, 5, 6) . In a number of animal studies, CpG motifs in bacterial DNA and synthetic ODN are responsible for driving immune responses toward Th1-type responses (7, 8, 9, 10) . The CpG sequences drive macrophages to secrete interleukin 12, a potent inducer of IFN-{gamma} production in vivo from natural killer cells. IFN-{gamma} production drives Th1-type immune responses through inducing differentiation of type 1 Th cells, which see antigen in the presence IFN-{gamma} from the uncommitted T-cell pool (7 , 11) . Moreover, ODN enhances humoral responses, driving them toward IgG2a isotypes (Th1 type indicator; Refs. 7 , 12 ) and induces development of enhanced CTL activity (3 , 13) . ODN has been studied extensively as strong immunomodulatory agents (12 , 14, 15, 16, 17) .

HPV 16 infection is a major cause of cervical cancer worldwide (18) . The expression of HPV oncogenic proteins, E6 and E7, is required for tumorigenesis and maintenance of the tumor state (19, 20, 21) . Furthermore, E7-specific immune responses are detected in cervical cancer patients (22) , suggesting that E7 could be a specific target for immunotherapy against HPV-derived cervical cancers. In this regard, E7-specific prophylactic and therapeutic vaccine strategies have been evaluated in animal model systems. These include direct uses of recombinant E7 proteins (23) , DNA vaccine encoding E7 (24) , and bacterial/viral vectors expressing E7 or E7 epitope (25, 26, 27, 28) , as well as CTL epitopes of E7 (29) . In these studies, CD4+ T-cell and in particular CTL activities have been correlated to protective immunity against tumor cells. However, no studies on effect of ODN for immunotherapy against cervical cancer have been reported.

In this study, we produced the recombinant E7 protein of HPV 16 type and tested its ability along with ODN to induce protection against E7-expressing tumors as well as E7-specific immune responses in an animal model system. We observe that injection with E7+ODN results in a significant suppression of tumor formation prophylactically and therapeutically. However, no such effect of E7 without ODN was observed. E7+ODN also enhanced E7-specific antibody (IgG1, IgG2a, IgG2b, and IgG3) and Th-cell proliferative responses significantly higher than E7 alone, suggesting an immune enhancing nature of ODN. In contrast, CTL and IFN-{gamma} production from CD4+ and CD8+ T cells were detected only in E7+ODN immunized groups. The tumor protection appears to be mediated by CD4+ and in most part CD8+ T cells, as determined by in vivo T-cell subset depletion. Taken together, these data suggest that E7+ODN codelivery could be a potent approach to drive Th1 type CD4+ T-cell and CTL activities for control of HPV-associated cervical cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Production of Recombinant E7 Protein.
The full protein coding region of the HPV 16 E7 gene was reverse transcription-PCR amplified from a CaSki cell line with a pair of primers: the BamHI containing sense primer, 5-TTGGGATCCACCATGCATGGAGATACACCTAC-3 and EcoRI-containing antisense primer, 5-CGGAATTCATTCTTATGGTTTCTG-3. The amplified DNA was digested with BamHI and EcoRI, and the resulting DNA fragment was gel purified. The E7 DNA fragments were then cloned into the BamHI and EcoRI site of the pET vector (Novagen, Madison, WI). The plasmid construct was transformed into Escherichia coli DH5{alpha} and selected against kanamycin. pET-E7 vector was purified and again transformed into BL21(DE3) cells, and incubated in Luria-Bertani broth supplemented with kanamycin at a final concentration of 30 µg/ml. The cells were incubated in a shaker until absorbance at 600 nm was between 0.6 and 0.8 absorbance units. Proteins were induced by addition of 1 mM isopropyl-1-thio-ß-D-galactopyranoside for 3 h. The cell pellets were collected at 4 krpm for 20 min and frozen-thawed once at -20°. The cell pellet was resuspended in 5 ml of 8 M urea buffer (pH 8.0) per gram wet weight. The cells were lysed by stirring for 15–60 min at room temperature and centrifuged at 1.5 krpm for 30 min. The cell supernatants were collected and passed through for the Ni-NTA resin column (Qiagen, Valencia, CA) pre-equilibrated with 8 M urea buffer (pH 8.0). The resin was washed with 5 vol of Buffer B [8 M urea buffer (pH 8.0)] and then with 5–10 vol of Buffer C [8 M urea buffer (pH 6.3)]. In the final step, His-tagged E7 protein was eluted with 10 ml of Buffer C containing 200 mM imidazole. The protein solution was then dialyzed in 6 M urea buffers and then in 4 M urea buffers at 2-h intervals. This was followed by overnight dialysis in PBS. The protein solution was collected and passed through the Detoxi-Gel endotoxin removing gel column (Pierce, Rockford, IL) according to the manufacturer’s protocol except for final elution with PBS. The protein concentration was calculated by the Bradford procedure using BSA as a standard (30) . The endotoxin level of the E7 recombinant protein was checked using the endotoxin detection kit (Sigma, St. Louis, MO). The final protein solution was stored at -70°C.

ODNs.
The immunostimulatory CpG ODN designated as 1826 (5'-TCCATGACGTTCCTGACGTT-3') was used as a vaccine adjuvant in this study. The ODN was purchased from Biobasic Inc., Toronto, Ontario, Canada. ODN was synthesized with a nuclease-resistant phosphorothioate backbone. ODN was dissolved in water and was confirmed to have undetectable endotoxin level.

SDS-PAGE and Immunoblot Analysis.
Protein samples were separated on 12% SDS polyacrylamide gel. The proteins were electrophoretically transferred to nitrocellulose membranes (Amersham, Piscataway, NJ). The membrane was pre-equilibrated with TBST solution [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20] containing 2% BSA for 1 h and then reacted with anti-E7 monoclonal antibodies (Oncogene, Boston, MA) for 1 h at room temperature. After three washes with TBST, the membrane was incubated with antimouse IgG-HRP (Sigma) for 1 h at room temperature. The immunoreactive protein bands were visualized using the ECL detection reagents (Amersham).

Immunization of Mice.
Female 4–6-week-old C57BL/6 mice were purchased from Daehan Biolink, Chungbuk, Korea. Mice were injected s.c. with 20 µg of recombinant E7 protein and/or 20 µg of ODN in a final volume of 100 µl of PBS using a 28-gauge needle (Becton Dickinson, Franklin Lakes, NJ).

ELISA.
ELISA was performed as described previously (31 , 32) . In particular, recombinant E7 protein (1 µg/ml in PBS) was used as a coating antigen. For the determination of relative levels of E7-specific IgG subclasses, antimurine IgG1, IgG2a, IgG2b, or IgG3 conjugated with HRP (Zymed, San Francisco, CA) were substituted for antimurine IgG-HRP. To determine ELISA titers, sera pooled in an equal volume from 10 mice per group were 2-fold serially diluted and reacted with E7 protein. The titers were determined as the reciprocals of the highest serum dilutions showing absorbance values twice as high as that of the negative control.

Th-Cell Proliferation Assay.
Th cell proliferation assay was performed as described previously (33 , 34) . In brief, spleen cells were stimulated with E7 proteins at 0.5, 1, and 5 µg/ml concentrations for 3 days. Then, [3H]-labeled thymidine (1 µCi/well) was added overnight. The next day, the cells were harvested, and cpm was counted using ß-counter (Perkin-Elmer, Boston, MA). Stimulation index was determined as [(experimental cpm - medium control cpm)/(media control cpm)].

Cytotoxic T-Lymphocyte Assay.
A 5-h 51Cr release assay was performed. Briefly, splenocytes were stimulated for 5 days in the presence of 20 units/ml of interleukin 2 (R&D Systems, Minneapolis, MN) with TC-1 cells treated previously for 3 h with mitomycin C (30 µg/ml). TC-1 target cells were labeled with 100 µCi/ml Na251CrO4 for 2 h and used to incubate the stimulated splenocytes for 5 h at 37°C. One-hundred µl of supernatants were harvested and counted on a gamma counter (Perkin-Elmer). The percentage of specific lysis was determined as 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Maximum release was determined by lysis of target cells in 1% Triton X-100. An assay was not considered valid if the value for the spontaneous release counts was in excess of 20% of the maximum release value.

IFN-{gamma} Assay.
A 1 ml aliquot containing 6 x 106 splenocytes was added to wells of 24-well plates. Then, 1 µg of recombinant E7 protein/ml or 4 x 105 TC-1 cells treated previously with mitomycin C (30 µg/ml for 3 h) was added to each well. After 3 days of incubation at 37°C in 5% CO2, cell supernatants were secured and then used for detecting levels of IFN-{gamma} using commercial cytokine kits (Biosource, Intl., Camarillo, CA) by adding the extracellular fluids to the IFN-{gamma}-specific ELISA plates.

FACS Analysis.
Spleen cells (1 x 105) were washed three times with FACS buffer (PBS +1% BSA +0.1% sodium azide) and then reacted with phycoerythrin-conjugated antimouse CD4 and CD8 (PharMingen, San Diego, CA) for 30 min on ice. After washing three times with FACS buffer, cells were analyzed for the percentage of CD4- or CD8-positive cells on a flow cytometer (Coulter-Epics XL, Miami, FL).

In Vitro and in Vivo Depletion of CD4+ and CD8+ T Cells.
Depletion studies were performed as described previously (33 , 34) . For in vitro cell depletion, splenocyes were reacted with anti-CD4 (PharMingen) or anti-CD8 (Accurate Chemical & Scientific Corp., Westbury, NY) for 1 h at 4°C, followed by incubation with rabbit complement (Sigma) for 1 h at 37°C. Cell viability after depletion was determined by trypan blue dye exclusion. Two cycles of antibodies plus complements resulted in depletion of >98% specific T-cell subpopulation by FACS analysis. For in vivo cell depletion, anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) ascites fluids were generated by injecting hybridoma cells (American Type Culture Collection, Manassas, VA) into pristane-primed nude mice i.p. One-hundred µl of ascites fluids were administered i.p. on days -3, 0, and 3 of tumor challenge. Antibody treatment resulted in >98% depletion of specific CD4+ and CD8+ T-cell subsets of representative animals over a 3-week period. Depleted mice were subsequently challenged with tumor on day 0.

Tumor Protection Assay.
Two x 105 and 5 x 104 TC-1 cells were injected s.c. into the right flank of C57BL/6 mice for prophylactic and therapeutic vaccine studies, respectively. These challenge doses were tested previously (35) . TC-1 tumor cells (a kind gift from T-C. Wu, Johns Hopkins Medical Institution, Baltimore, MD) were grown in complete RPMI supplemented with 400 µg/ml of G418. The tumor cells were washed twice with PBS and injected into mice. For therapeutic studies, at the time (1 week) of tumor size showing 1–2 mm after tumor challenge each group of mice was injected s.c. with E7 and/or ODN and then reinjected 1 week after the first injection. Mice were monitored twice per week for tumor growth. Tumor growth was measured in mm using a caliper, and was recorded as mean diameter [longest surface length (a) and width (b), (a+b)/2]. Mice were euthanized when the tumor size reached >20 mm in mean diameter.

Statistical Analysis.
Statistical analysis was done using the paired Student’s t test. Values of E7 injection alone were compared with values of E7+ODN coinjection groups. Ps < 0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Recombinant E7 Protein.
The recombinant E7 protein encompassing 98 amino acid residues of the full-length E7 gene was expressed and purified in E. coli. We were able to obtain 4 mg of the recombinant protein from 1000 ml of bacterial cell culture. Endotoxin levels of the recombinant protein were determined to be <100 EU/mg. The recombinant E7 protein migrated as a 23 kDa protein in SDS-PAGE and was reactive to the HPV 16 E7 monoclonal antibodies (data not shown). A molecular mass of the 23 kDa protein was larger in size than predicted (11 kDa of E7 protein plus 4 kDa protein of His-tagged regions in the pET vector system). This abnormal migration pattern of E7 protein was reported previously (23 , 36) .

E7+ODN Injection Resulted in Complete Protection from E7-expressing Tumor Challenge.
It is important that antigen-specific immune modulation influences tumor formation. We analyzed protective efficacy of ODN coinjection in the murine tumor challenge model. As shown in Table 1Citation and Fig. 1Citation , mice were immunized with E7 (20 µg/mouse) and/or ODN (20 µg/mouse), and then challenged with 2 x 105 of TC-1 cells. TC-1 is an E7-expressing tumorigenic cell line. It was established from primary lung epithelial cells of C57BL/6 mice immortalized with HPV 16 E6 and E7, and then transformed with an activated ras oncogene (37) . This cell line has been used extensively for tumor challenge studies (24, 25, 26, 27 , 35) . E7+ODN injection alone resulted in complete protection from tumor challenge, whereas E7 or ODN vaccine alone showed 100% tumor formation in animals in a manner similar to negative controls. Fig. 1Citation shows one representative experiment. Mice given injections of TC-1 cells developed rapidly growing tumors at the site of injection in negative control, E7, and ODN-immunized animals over time. However, no tumor growth was observed in mice given injections of E7+ODN. This illustrated that ODN as a vaccine adjuvant induced complete protection from tumor challenge. In particular, E7 or ODN injected groups, as well as negative group animals were all dead within 30–40 days after tumor challenge, whereas E7+ODN group survived far longer than 45 days (>4 months). Postmortem autopsy confirmed that death was because of cachectic shock and showed no signs of metastasis to other organs.


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  		Table 1 Number of mice with tumor/number mice challenged with tumor (%)

Each group of mice (n = 11–16) was immunized s.c. with 20 µg of E7 and/or 20 µg of ODN at 0 and 2 weeks. Three weeks after the second injection, mice were challenged s.c. with 2 x 105 TC-1 cells. The number of animals showing tumor was counted as described in "Materials and Methods."

 


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  		Fig. 1. Effects of E7+ODN codelivery on tumor cell growth over time. Each group of mice (n = 6) was immunized s.c. with 20 µg of E7 and/or 20 µg of ODN at 0 and 2 weeks. Three weeks after the second injection, mice were challenged s.c. with 2 x 105 TC-1 cells. Tumor size was monitored as described in "Materials and Methods," and then its mean diameter was plotted over time.

 
E7+ODN Injection Therapeutically Protected Animals from Tumor Formation.
Next we sought to determine whether E7+ODN injection could confer protection to animals with established tumor. Animals challenged previously with TC-1 cells were injected twice with E7 and/or ODN, and then observed for tumor growth for 1 month after tumor challenge. As shown in Fig. 2Citation , the groups injected with E7+ODN had 20% animals exhibiting tumor on the flank. In contrast, mice immunized with E7 or ODN alone showed 100% animals with tumor similar to negative control. Mice showing about 1–2 mm in tumor size developed rapidly growing tumors at the site of injection over time when immunized with E7 or ODN. However, tumor growth was suppressed complete in mice given injections of E7+ODN with the exception of 2 of 10. These 2 mice displayed a more slowly growing tumor, as compared with other control groups. Furthermore, E7 or ODN injected groups, as well as negative control animals were all dead within 40 days after tumor challenge, whereas E7+ODN-injected animals survived far longer than 2 months. This supports that E7+ODN coinjection can induce protection from established tumor.



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  		Fig. 2. Therapeutic effects of injection with E7 and/or ODN on tumor growth. Each group of mice (n = 10) was challenged s.c. with 5 x 104 TC-1 cells. When tumor size reaches 1–2 mm, animals were injected s.c. to the distal site of tumor with 20 µg of E7 and/or 20 µg of ODN. The second injection was followed at 1 week after the first injection. Animals were evaluated for regression of tumor two times every week.

 
E7+ODN Vaccination Enhanced Systemic IgG and IgG Subclass Production.
We first investigated the in vivo effects of E7 and E7+ODN on the induction of antigen-specific antibody responses. As shown in Fig. 3ACitation , ELISA titers of equally pooled sera collected 2 weeks after the second immunization were determined as 1600 (E7) and 6400 (E7+ODN), a 2-fold increase in titer. In similar, those of sera collected 6 weeks after the second immunization were determined to be 800 (E7) and 3200 (E7+ODN). However, little induction of antibody titer was observed in ODN-injected groups similar to negative control. We also observed the production pattern of IgG subclasses, as they give an indication of the Th1 versus Th2 nature of the induced immune responses. It has been known that IgG1 and IgE are Th2-associated antibodies, whereas IgG2a is a Th1-associated isotype antibody (38) . We analyzed the IgG subclasses induced by the ODN coinjection. As shown in Fig. 3, B–ECitation , E7+ODN vaccination enhanced all four types of IgG isotype production significantly higher than E7 vaccination alone. This pattern was observed 4 and 8 weeks after the first immunization. In particular, IgG2a production was significant by E7+ODN injection, as compared with E7 injection alone. IgG2a/IgG1 was calculated as 0.2 (E7) and 0.26 (E7+ODN). This analysis suggests that ODN drives humoral immune responses overall in vivo.



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  		Fig. 3. Induction of E7-specific IgG and IgG isotypes by injection with E7 and/or ODN. Each group of mice (n = 10) was immunized s.c. with 20 µg of E7 and/or 20 µg of ODN at 0 and 2 weeks. Mice were bled at 2, 4, and 8 weeks after the first injection. A, equally pooled 2-, 4-, and 8-week sera were serially diluted, and reacted with E7 to determine ELISA titers. B–E, equally pooled 4- and 8-week sera/group were diluted to 1:100 and reacted with E7 protein in ELISA. Absorbance was measured at 405 nm. *, statistically significant at P < 0.05 using Student’s t test compared with E7 alone.

 
E7+ODN Enhanced Th-Cell Proliferative Responses.
Th-cell proliferation is a standard parameter used to evaluate the potency of cell-mediated immunity. We measured Th-cell proliferative responses after coimmunization with ODN by stimulating splenocytes from immunized animals in vitro with E7 proteins. As shown in Fig. 4ACitation , E7 vaccination resulted in E7-specific Th-cell proliferative responses. We also observed the significant enhancement of Th-cell proliferative responses over that of E7 vaccine alone by immunization with E7+ODN. In contrast, negative control group and ODN-immunized group showed little effects on the levels of Th-cell proliferative responses.



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  		Fig. 4. Induction of E7-specific Th-cell proliferative and CTL responses by injection with E7 and/or ODN. Each group of mice (n = 4) was immunized s.c. with 20 µg of E7 and/or 20 µg of ODN at 0 and 2 weeks. Three weeks after the last immunization, spleen cells were obtained. For Th cell proliferation assay (A), splenocytes were stimulated in vitro with 0.5, 1, and 5 µg/ml E7 proteins. After 3 days of stimulation, cells were harvested and then cpm was counted. Samples were assayed in triplicate. For CTL assay (B), splenocytes were stimulated in vitro with mitomycin C-treated TC-1 cells. The specific cytolytic activity was tested against TC-1 cells in a 51Cr release assay. Results represent mean specific lysis values from individual, representative mice tested at the indicated E:T cell ratio. The experiments were repeated two more times with similar results. *, statistically significant at P < 0.05 using the paired Student’s t test compared with negative controls. **, statistically significant at P < 0.05 using the paired Student’s t test compared with E7 alone; bars, ±SD.

 
CTL Was Induced by Immunization of E7+ODN Alone.
To determine whether E7+ODN could induce E7-specific CTL activity in vivo, we immunized animals with E7 and/or ODN. As shown in Fig. 4BCitation , injection with E7 plus ODN alone induced CTL activities to a significant level. However, animals immunized with E7 or ODN alone showed little induction of CTL. This finding suggests that E7 in the presence of ODN can induce E7-specific CTL responses in vivo.

IFN-{gamma} Production from CD4+ T Cells by Injection with E7+ODN.
IFN-{gamma} has been known to play an important role in driving Th1-type immune responses as well as inducing cytotoxic T-cell responses. To evaluate the CD4+ T cell-dependent production level of IFN-{gamma}, we stimulated immune cells of immunized animals with E7 proteins. As shown in Fig. 5ACitation , IFN-{gamma} production was induced dramatically by injection with E7+ODN. However, little induction of IFN-{gamma} production was observed by injecting E7 or ODN alone. This illustrates that E7 in the presence of ODN drives T-cell responses predominantly in a Th1-type fashion. When splenocytes of E7+ODN-immunized animals were depleted of CD4+ T cells, IFN-{gamma} production was decreased to a background level, whereas CD8+ T-cell depletion resulted in the same enhancement of IFN-{gamma} production as whole splenocytes from E7+ODN injected animals (Fig. 5B)Citation , suggesting that CD4+ T cells are responsible for enhanced Th1-type cellular responses through injection of E7+ODN.



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  		Fig. 5. Production levels of IFN-{gamma} from splenocytes in mice immunized with E7 and/or ODN. Animals were immunized as shown in Fig. 4Citation . Three weeks after the last injection, mice were sacrificed and spleen cells were pooled. Splenocytes were stimulated with 1 µg of E7 proteins/ml for 3 days (A). Splenocytes of E7+ODN- immunized animals were depleted in vitro of either CD4+ or CD8+ T cells, and then stimulated with 1 µg of E7 proteins/ml for 3 days (B). Splenocytes were stimulated with mitomycin C-treated TC-1 cells for 3 days (C). Splenocytes of E7+ODN-immunized animals were depleted in vitro of either CD4+ or CD8+ T cells and then stimulated with mitomycin C-treated TC-1 cells for 3 days (D). Samples were assayed in triplicate. Values represent mean of released IFN-{gamma} concentrations; bars, ±SD. The experiments were repeated two more times with similar results.

 
IFN-{gamma} Production from CD8+ T Cells by Injection with E7+ODN.
We were next interested in evaluating the CD8+ T cell-dependent production level of IFN-{gamma}. Splenocytes of animals immunized with E7 and/or ODN were subsequently stimulated in vitro with E7-expressing syngeneic TC-1 cells (MHC class I+, class II-). As shown in Fig. 5CCitation , IFN-{gamma} production was dramatically induced by injection with E7+ODN. However, little induction of IFN-{gamma} production was observed in the groups injected with E7 or ODN alone. This is consistent with our previous observation that CTL (IFN-{gamma}-secreting CD8+ T cells) was induced only by E7+ODN coinjection. Furthermore, when splenocytes of E7+ODN-immunized animals were depleted of CD8+ T cells, a background level of IFN-{gamma} production was detected, in contrast to depletion of CD4+ T cells (Fig. 5D)Citation , supporting the idea that CD8+ T cells are responsible for IFN-{gamma} production in a MHC class I-dependent manner.

Tumor Protection Was Mediated by CD4+ and in Most Part CD8+ T Cells in Vivo.
We next focused on possible roles of CD4+ or CD8+ T cells in inducing protective immunity induced by E7+ODN against challenge with E7-expressing TC-1 tumor cells. As shown in Fig. 6Citation , after E7+ODN vaccination we depleted CD4+ and CD8+ T cells or both in vivo, and then tested effects of specific cell populations on tumor protection. When animals immunized previously with E7+ODN were challenged with tumor cells, complete protection from tumor formation was observed in the absence of immune cell depletion. However, animals depleted of both CD4+ and CD8+ T cells failed to protect tumor growth in a manner similar to negative control animals. In particular, CD8+ T cell-depleted animal group showed a bit delayed but complete formation of tumor, as compared with a negative control group or the animal group depleted of both CD4+ and CD8+ T cells, suggesting a contributing role of CD4+ T cells and a major role of CD8+ T cells in protection against tumor formation. Moreover, animals depleted of CD4+ T cells protected tumor growth in 4 of 5 animals. In particular, the remaining 1 CD4+ T cell depleted animal displayed a far smaller tumor than other groups with tumors over the time periods (data not shown). Taken together, these data support that E7 in the presence of ODN can induce protection from tumor growth through effects on CD4+ T cells and in most part CD8+ T cells in vivo.



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  		Fig. 6. Effects of T-cell subsets on tumor growth. Each group of mice (n = 5) was immunized s.c. with 20 µg of E7 and/or 20 µg of ODN at 0 and 2 weeks. Three weeks after the second injection, mice were depleted in vivo of CD4+ and CD8+ T cells, or both as described in "Materials and Methods." Animals were challenged s.c. with 2 x 105 TC-1 cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we observed that coinjection with ODN plus E7 induced a complete protection of animals from subsequent HPV 16 E7-associated tumor challenge. However, injection with either E7 or ODN failed to show any protection from tumor growth. This was also true in therapeutic effects in which tumor growth was suppressed only by E7+ODN codelivery. These suggest that both E7 and ODN are required for prophylactic and therapeutic vaccine efficacies in this tumor model system. However, in this case, antitumor adjuvant effects of ODN might be ascribed at least in part to a phosphorothioate ODN backbone sequence itself not along with CpG motifs. However, our observation is in line with previous findings that coinjection with tumor antigen and CpG DNA sequence provides more protection from tumor challenge in a murine B-cell lymphoma model (14 , 39) . More recent literature also showed that intranasal delivery of herpes simplex virus (HSV)-1 gB protein plus ODN enhances protection against intravaginal HSV-2 challenge, as compared with gB alone (17) . Furthermore, ODN itself or as an adjuvant enhances protection of animals from bacterial infection (40 , 41) . However, in our case, antigen or ODN alone showed no beneficial effects on tumor regression. This difference might be because of antigen types tested, the doses of antigen or ODN used, or injection routes tested.

Previous literature has shown positive effects of ODN on immune responses. For example, coinjection with hepatitis B surface antigen plus CpG DNA sequence results in enhanced antigen-specific humoral, in particular IgG2a and CTL responses (12) . In the present studies, coinjection with E7 plus ODN enhanced systemic IgG production significantly higher than E7 vaccination alone. This is compatible with previous findings of other groups (12 , 14 , 17 , 39) . Furthermore, there was also a significantly enhanced production of all four IgG isotypes, IgG1, IgG2a, IgG2b, and IgG3 by ODN coinjection. This is supported by previous findings that both hepatitis B surface antigen-specific IgG1 and IgG2a types were significantly enhanced by coinjection with ODN (12) . Similarly, Th-cell proliferative responses were enhanced by coinjection with E7 plus ODN significantly higher than E7 injection alone in a manner similar to antibody responses we observed. In this case, Th-cell proliferation was not observed when immune cells were depleted of CD4+ T cells (data not shown). This additionally suggests that antibody production levels correlate well with CD4+-dependent Th-cell proliferation levels.

We also evaluated CTL activities when antigen was delivered along with ODN. CTL was significantly induced by coinjection with E7+ODN alone. In contrast, there was little induction of CTL activities by delivery of either E7 or ODN alone. This is supported by previous findings that antigen-specific CTL activity is induced by codelivery with the antigens of HSV-1 or HBV plus ODN (12 , 17) , supporting that ODN codelivery could be useful for induction of antigen-specific CTL activities. Involvement of ODN for production of IFN-{gamma} has been reported previously (7) . We also observed that on in vitro stimulation with E7 there was an induction of IFN-{gamma} production from CD4+ T cells in the E7+ODN injected group, as compared with E7- or ODN-injected group. This suggests that on antigen stimulation, CD4+ T cells but not CD8+ T cells are responsible for secretion of IFN-{gamma}. In similar, we observed that on in vitro stimulation with TC-1 cells there was an induction of IFN-{gamma} production from CD8+ T cells in E7+ODN-injected group, as compared with E7- or ODN-injected group. This additionally confirms that on TC-1 tumor cell stimulation, CD8+ T cells but not CD4+ T cells are responsible for secretion of IFN-{gamma}. This correlates well with CTL induction by E7+ODN injection we observed. Thus, these collective data support the idea that both E7 and ODN are required for induction of IFN-{gamma} from CD4+ and CD8+ T cells in a MHC-specific fashion. Moreover, the protective role of IFN-{gamma} against virus infection or tumor challenge has been reported previously (42, 43, 44, 45) . Taken together, ODN can be used as a cervical cancer vaccine adjuvant to direct E7-specific immune responses to both IFN-{gamma}-secreting CD4+ T cell (Th1) and IFN-{gamma}-secreting CD8+ T cell (CTL) types in vivo.

We additionally evaluated a possible role of CD4+ versus CD8+ T cells for protecting animals from the TC-1 tumor challenge. When animals were depleted of both CD4+ and CD8+ T cells, tumor growth was observed in a manner similar to negative control group. However, CD8+ T cell-depleted animal group showed a bit delayed but complete loss of protection against tumor, suggesting CD8+ T cells are a major effector T-cell population. When CD4+ T cells were depleted, protection from tumor was observed in 4 of 5 animals, as compared with 100% protection of the nondepleted animal group. These suggest that CD4+ and in most part CD8+ T cells play a critical role in protection against TC-1 tumor. However, in our observation, antibody responses appeared not to be directly correlated with protective immunity against TC-1 tumor (Table 2)Citation . Furthermore, previous literature has demonstrated that CD4+ and/or CD8+ effector T cells play a major role in protecting animals from challenge with TC-1 cells (24, 25, 26, 27 , 35) .


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  		Table 2 Immune responses and protection against tumor challenge

 
In conclusion, we observe that codelivery with E7+ODN could be useful for induction of protective immunity against HPV-associated tumor cells through induction of antigen-specific Th1 type CD4+ and in most part CD8+ T-cell responses in animals. This study likely provides an additional option for inducing E7-specific T cell-mediated immune responses as a possible immune therapy modality against HPV-associated cervical cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. T-C. Wu for providing TC-1 cells. J-I. S. thanks Su-Mi Bae for technical assistance in this study and Dr. D. B. Weiner for his helpful advice.


   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.

1 Supported by Korea Research Foundation Grant (KRF-2001-041-F00085). Back

2 To whom requests for reprints should be addressed, at 6004-2, Cancer Research Center, The Catholic Research Institutes of Medical Science, 505, Banpo-Dong, Seocho-Ku, Seoul 137-040, Korea. Phone: 82-2-590-2405; Fax: 82-2-599-4120; E-mail: jsin1964{at}hanmail.net Back

3 The abbreviations used are: ODN, oligodeoxynucleotide; HPV, human papillomavirus; HRP, horseradish peroxidase; HSV, herpes simplex virus; Th, T-helper; FACS, fluorescence-activated cell sorter. Back

Received 7/ 5/02. Accepted 10/10/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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