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Immunology |
Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois 60153 [G. L. E., M. P. V., W. M. K.]; Department of Pathology, University of Chicago, Chicago, Illinois 60637 [H. S.]; and Wyeth Research, Viral Vaccines Immunology, Pearl River, New York 10965 [M. C. C., J. K. P., L. R. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many HPV vaccine strategies have successfully prevented tumor growth upon challenge with HPV16-positive tumor cells in C57BL/6 mice and have elicited an H-2-restricted immune response against the H-2Db HPV16 E7-restricted epitope, RAHYNIVTF (49–57). The vaccination strategies have included viral vectors (11 , 12) , synthetic peptides (13) , recombinant proteins (14, 15, 16) , chimeric virus like particles (17, 18, 19) , and plasmid DNA (20 , 21) . Unfortunately, DNA vaccination with intact E6 or E7 genes has a risk of oncogenic transformation through integration of recombinant genes into the host genome, whereas peptide vaccines are limited to predetermined target HLA alleles and could possibly induce T-cell tolerance (22) . Potential drawbacks from the use of chimeric virus-like particles or viral vector systems include the induction of neutralizing antibodies against viral structural proteins in preimmune patients or after subsequent vaccination (23) . The use of an alphavirus delivery vector may eliminate most of these drawbacks. Alphaviruses such as VEE virus replicate the RNA of interest in the cell cytosol and are cytopathic, thereby significantly reducing the risk of integration of E6and E7into the cellular genome. By infecting dendritic cells, VRPs target expression to lymphoid tissue, a preferred site for the induction of immunity (24) . Furthermore, repeated immunizations may be possible because there is no widespread existing immunity to VEE in humans. Recently, we have reported that E7-VRP vaccination induced CTLs capable of complete tumor protection and elimination of 67% of established tumors in C57BL/6 mice (12) .
Another promising vaccination strategy is a multi-epitope plasmid DNA vaccine encoding several CTL epitopes from multiple HLA types, rather than entire proteins (20) . This strategy eliminates any possible risk of oncogenic transformation and the downfall of targeting one HLA restriction element. We have reported on the construction of several DNA based multi-epitope vaccines containing a combination of HPV16 CTL, T-helper cell, and B-cell epitopes (20) . Gene gun-mediated delivery of these DNA constructs induced protective immune responses and cured established tumors in H-2Db mice (21) .
Although both VRP and multi-epitope pDNA vaccination strategies show encouraging results, H-2b-restricted T-cell recognition of HPV antigens in C57BL/6 mice provides limited predictive value for HLA restricted antitumor responses. Therefore, the demonstration that the same vaccine can induce HLA-A*0201-restricted responses against HPV16 E6 and E7 in HLA-A*0201 transgenic mice and humans is particularly important (10) . CD8+ T cells from HLA-A*0201 transgenic mice have been shown to recognize the same HLA-A*0201-restricted antigens as those recognized by HLA-A*0201-restricted human CTLs (25 , 26) . The use of HLA transgenic mice could therefore overcome the limitations of H-2-restricted tumor rejection. However, no HPV tumor model has been available to test HLA-A*0201-restricted antitumor responses. Here we present the first HPV16 tumor model for HLA-A*0201 transgenic mice. Furthermore, we have tested the vaccination with HPV16 peptides, E7-E6 VRPs, and our multi-epitope DNA vaccine for the processing and presentation of HLA-A*0201-specific epitopes and induction of HLA-A*0201-restricted protective and therapeutic antitumor immune responses.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 domain of the heavy chain replaced by the corresponding murine H2 Dd 3 domain, whereas the 1 and 2 domains are of human HLA-A*0201 origin. This allows for the interaction of the murine CD8 molecule with the murine 3 domain, whereas the peptide-binding characteristics associated with human 1 and 2 domains are retained. Mice were housed in the animal facilities of Loyola University Chicago under filtertop conditions with water and food ad libitum. Institutional animal use guidelines were followed for all experiments.
YAC-1, EL4, and EL4 A2Kb cells were used for cytotoxicity assays and were cultured in IMDM (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FCS (JRH Biosciences, Lenexa, KS), 2 mM L-glutamine, 100 µg/ml kanamycin, and 50 mM 2-mercaptoethanol. EL4 A2Kb is a transfectant of the EL4 thymoma that expresses the 1 and 2 domains of HLA-A*0201 in association with the 3 domain of H2-Kb. This line was maintained under G418 sulfate (Geneticin) selection (500 µg/ml).
The HLF16 tumor cell line was established by removing heart and lung tissue from HLA-A2 Dd mice and growing the tissue in IMDM for 6 weeks. Surviving fibroblasts were then transformed with a geneticin resistance-conferring, bicistronic pIRES vector (Clontech, Palo Alto, CA) encoding E6, E7 (-49–57) and H-ras, Q61L. Transformed colonies were selected with 2 mg/ml G418, and single clones were picked and expanded. Clones were then treated with 100 units/ml IFN-
for 3 days and screened by fluorescence-activated cell sorting analysis with HLA-A*0201-specific antibody BB7.2 for HLA-A2 expression, and three clones were selected to test in the soft agar assay.
Soft Agar Assay.
Anchorage-independent growth capability was determined by assessing the colony formation efficiency of cells suspended in soft agar. Transformed and control cells (0.5 x 106, 0.25 x 106, and 0.1 x 106) were seeded in 5 ml of 0.3% overlay agar and added to 10-mm plates coated with 20 ml of 0.6% underlay agar. Plates were allowed to dry and were incubated at 37°C. Colonies were counted 3 weeks after plating.
Immunofluorescence Staining.
E7-specific protein expression in the HLF16 cell line was confirmed by immunofluorescence as described previously (12)
. HLF16 and HLF untransfected controls were fixed with methanol/acetone and incubated with a mouse monoclonal anti-E7 antibody (Zymed, San Francisco, CA), washed, and stained with goat antimouse immunoglobulin FITC and mounted in Vectrashield containing 4',6-diamidino-2-phenylindole. E7-positive cells were visualized with a fluorescent microscope.
DNA Vaccine Construction.
The selection of epitopes represented in the current DNA vaccine is based on the previous identification of HPV16 epitopes for HLA-A*0201 and HLA-A*2401 (27)
. The vaccine was constructed as described previously (21)
with alanine-alanine-tyrosine (AAY) spacers between epitopes and an ubiquitin molecule at the COOH terminus. The H-2Db dominant HPV16 E749–57 epitope was removed by recombinant PCR to prevent an H-2Db-restricted immune response. Primers were prepared to amplify the 5' and 3' surrounding RAHYNIVTF with overlapping primers. DNA was then isolated, and single-stranded regions were fused by 2.5 units of PFU polymerase (Stratagene, La Jolla, CA) in the following recombinant PCR reaction: one cycle for 4 min at 94°C, 25 cycles for 2 min at 94°C, plus 4 min at 72°C. The double-stranded DNA was amplified with 5' and 3' primers in a standard PCR reaction using PFU polymerase (Stratagene). The PCR product was then cloned into the EcoRV site of pZero (Invitrogen, Carlsbad, CA) and subcloned using BamHI-ApaI into the mammalian expression vector, pcDNA3 (Invitrogen) for vaccination.
DNA-Gold Bead Preparation and Gene Gun-mediated Delivery.
The Helios gene gun system (Bio-Rad, Hercules, CA) was used for epidermal gene delivery. Gold beads containing 2 µg of DNA/shot were generated using the manufacturer’s protocol and as described previously (21)
. The bullet-containing cartridges were loaded into the gene gun and delivered into the mouse abdominal epidermis at a helium pressure of 450 psi.
Preparation of VRPs.
A HPV16 E7-E6 gene fusion encoding 98 amino acids of E7 and 150 amino acids of E6 was configured as a single open reading frame. The 
750-bp E7-E6 open reading frame was subcloned into the VEE replicon plasmid, pVR200 (a proprietary vector from Alphavax, Durham, NC). PVR200 was derived from the cDNA of a highly attenuated, nonneurotropic mutant (V3014) of the Trinidad donkey strain of VEE. Replication-incompetent VRPs were prepared by the split helper method and titrated as described previously (12)
. VRP titers were expressed as infectious units per milliliter (IU/ml) in reference to the number of E7-E6-expressing viral particles.
Tumor Protection and Therapeutic Experiments.
For multi-epitope DNA vaccination, groups of 8 HLA-A*0201 transgenic mice were anesthetized by i.p. injection of 2.4 mg of ketamine (Abbott Laboratories, Chicago, IL) mixed in 80 µl of PBS with 0.48 mg of xylazine (Sigma Chemical Co., St. Louis, MO). The abdominal area was shaved, and the DNA was delivered into the epidermis via the gene gun. This procedure was repeated 2 weeks after the first DNA delivery and 9 days after the second vaccination mice were challenged s.c. with 2 x 106 HLF16 tumor cells in 100 µl of HBSS (Sigma). Tumor development was then monitored three times/week.
For VRP vaccination, groups of 8 HLA-A*0201 mice were vaccinated with 3 x 105 infectious units of E7-E6VRP s.c. in the left flank and boosted with the same dose 2 weeks later. Nine days after the booster dose, mice were challenged s.c. in the right flank with 2 x 106 HLF16 tumor cells in 100 µl of HBSS (Sigma).
For therapeutic experiments, mice first received a s.c. injection of 2 x 106 HLF16 cells in the right flank. At day 5, all of the mice had developed palpable tumors and received either the E7-E6VRPs or the multi-epitope vaccine. Vaccinations were repeated at day 10 and 15 days after the first vaccination. Tumor sizes in the mice were recorded two to three times/week.
Cytoxicity Assay.
Assays were performed 2 weeks after immunizations and subsequent HLF16 tumor challenge. Splenocytes were restimulated in vitro (30:1) with irradiated HLF16 tumor cells in a 24-well plate for 5–6 days. EL4 A2Kb cells loaded with either an irrelevant HLA-A*0201 PSA1 peptide (28)
, HPV16 E629–38, HPV16 E711–20, or E786–93 and HLF16 cells served as targets. EL4 cells loaded with HPV16 E749–57 also served as controls. Cells were incubated for 2 h with 20 µg/ml peptide at 37°C. Target cells were then labeled with 50 µCi of chromium (Sigma) for 90 min. Effector:target cells were added at the indicated ratios, and YAC-1 cells were added at a 10:1 ratio to the targets. Plates were incubated for 4 h at 37°C and 5% CO2. The percentage of specific lysis was calculated as:
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ELISPOT Assay.
An ELISPOT assay was used to detect peptide-specific T cells after stimulation with the synthetic HPV16 HLA-A*0201 peptides. Splenocytes were stimulated in vitro after two vaccinations with either the E7-E6VRPs or the multi-epitope DNA construct. Splenocytes were added to 24-well plates coated with irradiated heart and lung fibroblasts from HLA-A*0201 mice that had been incubated with either E629–38, E711–20, or E786–93 peptides overnight. Multiscreen HA plates (Millipore, Bedford, MA) were coated with 5 µg/ml anti-IFN- antibody (PharMingen, San Diego, CA) at 37°C overnight. Plates were washed with PBS/0.5% Tween and blocked with culture medium. Splenocytes were harvested and added at 1 x 106 and diluted 2-fold. After 24 h incubation at 37°C and 5% CO2, plates were washed with PBS/0.5% Tween and tap water and were incubated with 5 µg/ml biotinylated anti-IFN- antibodies (PharMingen) overnight at 4°C. After washing with PBS/0.5% Tween, 1.25 µg/ml avidin alkaline phosphatase (Sigma) was added to the well in 100 µl of PBS for 1 h at room temperature. The development of the assay was performed with 100 µl of 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (tablets; Sigma) for 10 min. The reaction was stopped by the addition of tap water, and the plates were allowed to dry before counting individual spots with a dissecting microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Clones that showed the highest HLA-A*0201 expression after IFN- treatment were subsequently tested for their ability to form colonies in soft agar (Fig. 1B)
. Heart and lung fibroblast clone 16 (HLF16) showed anchorage-independent growth in soft agar and was chosen for further studies. E7 expression was evident in the cytoplasm and nucleus of HLF16 after immunofluorescence staining with an anti-E7 monoclonal antibody (Fig. 1C)
. To determine whether the HLF16 line would in fact form tumors in mice, HLA-A*0201 transgenic mice were injected with different concentrations of tumor cells and monitored for 35 days. All mice developed tumors, but only those challenged with the highest dose, 2 x 106 HLF16 cells, maintained a tumor over the time course (Fig. 1D)
. The HLF16 line formed noninvasive, vascularized, s.c. tumors that arose at approximately day 5 and continued to grow progressively until it became approximately 12 x 12 x 12 mm by day 35, at which point the mice were sacrificed.
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. All mice vaccinated with irradiated HLF16 tumor cells were also completely protected against tumor outgrowth (data not shown). Complete protection was a reproducible finding in three independent experiments (data not shown).
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. Peptide-specific CTL responses were not detectable in the naïve mice. E7-E6VRP vaccination consistently showed the greatest lysis for the E786–93 epitope and the least for the E629–38 epitope (Fig. 3A)
, whereas the multi-epitope DNA vaccination resulted in the greatest lysis for the E711–20 epitope (Fig. 3B)
. CTL responses in E7-E6VRP vaccinated mice were significantly lower against the irrelevant PSA1 peptide. No Db-restricted CTL response against HPV16 E749–57 could be demonstrated, thereby confirming the removal of this dominant epitope from the multi-epitope vaccine. In contrast, a CTL response was generated against RAHYNIVTF in E7-E6VRP vaccinated mice because this construct encodes a fusion protein containing the Db epitope. However, this response is not involved in tumor rejection because this particular epitope was removed from the HLF16 tumor. This is demonstrated by the absence of recognition of the tumor cells by a RAHYNIVTF-specific CD8+ T-cell clone (data not shown).
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release after stimulation of splenocytes with either E711–20, 86–93 or E629–38 peptides. Splenocytes from vaccinated HLA-A*0201 transgenic mice were stimulated overnight with fibroblasts from HLA-A*0201 mice loaded with one of the peptides or a control PSA1 peptide. Peptide-specific responses were not observed for naïve mice or against the control PSA1 peptide. E7-E6VRP vaccination induced IFN- secretion in response to all HLA-A*0201 peptides, with a slightly better response against the E629–38 peptide (Fig. 4A)
. The multi-epitope DNA vaccination induced the best response against the E711–20 epitope (Fig. 4B)
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reveals that four of the five mice receiving E7-E6VRP rejected their tumors, whereas two of the five mice receiving the multi-epitope DNA vaccine rejected their tumors. The eradication of tumors was reflected in the mean tumor sizes of the groups (Fig. 5B)
. These data clearly show that although therapeutic vaccination did not always eradicate the tumor completely, it did significantly reduce its growth as compared with the controls. A second experiment gave comparable results (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have used this new tumor model to characterize both the immunological and the antitumor responses to HLA-A*0201-specific epitopes after treatment with two different HPV16-specific vaccines. The first vaccine is an extensive multi-epitope DNA construct that delivers relevant HPV16 E6 and E7 CTL epitopes while avoiding the possibility of transformation by integration of full-length E6 and E7 DNA. The second vaccine uses a replication-incompetent alphavirus to deliver RNA encoding the entire HPV16 E6 and E7 proteins. This allows for the processing and presentation of all HPV epitopes, including B-cell, T helper and CTL epitopes for every haplotype, while minimizing the risk of transformation. Our current studies have shown that vaccination with both the alphavirus containing HPV16 E7-E6 RNA and the HPV16 multi-epitope DNA vaccine are capable of protecting 100% of HLA-A*0201 mice from a challenge with the HLF16 tumor (Fig. 2)
. Moreover, vaccination elicited HLA-A*0201-restricted CTL responses against HPV16 that significantly reduced the growth of established tumors. We show that E7-E6VRP vaccination 5 days after HLF16 tumor challenge caused tumor regression in 80% of mice after three weekly injections (Fig. 5)
. Although the multi-epitope DNA vaccine was only capable of eradicating established tumors from 40% of the mice, a significant reduction in tumor burden was observed compared with the unvaccinated mice (Fig. 5B)
. These data indicate that both vaccination strategies were capable of inducing T cells responsible for antitumor responses in the presence of a growing tumor. Furthermore, because the tumor cell did not contain the only known HPV16 E7 H-2Db epitope, the eradication of these established tumors was mediated by an HLA-A*0201-restricted CTL response.
Our in vitro immune assays demonstrate that vaccination with E7-E6VRP and the multi-epitope plasmid DNA elicit MHC class I-restricted CD8+ T-cell responses against established HLA-A*0201 CTL epitopes (Fig. 3)
. Peptides E711–20, E786–93, and E629–38 are processed and presented in HLA-A*0201 mice and can induce CTL responses as well as peptide-specific release of IFN-. CTL responses against E711–20, E786–93, and E629–38 have been detected previously in individuals with cervical carcinoma or intraepithelial neoplasia, indicating that these peptides represent naturally processed human CTL epitopes of HPV16 (31
, 32)
. Our studies confirm these findings in a transgenic mouse model and for the first time demonstrate that these HLA-A*0201-specific CTLs are also capable of lysing tumor cells in vivo.
The tumor rejection and therapy experiments were performed in full-length HLA-A*0201 transgenic mice challenged with the chimeric HLA-A2Dd-expressing tumors. The chimeric A2Dd and the full-length HLA-A*0201 differ in their 3 domain, which is involved in the interaction with the CD8 molecule on the T cell but are not directly involved in peptide binding. It has been reported previously that a murine 3 domain enhances the interaction between the murine CD8 and the chimeric MHC molecule (33)
and thus would be preferential for mounting an immune response. Here we show that a HLA-A*0201-positive tumor, which contains the murine 3 domain, can develop in HLA-A*0201 mice lacking a murine 3 domain and that these tumors can respond to vaccination and therapy. A possible explanation for this observation is that a strong interaction with the 3 domain is not required for priming of the CD8 T-cell response through vaccination but that it is required, or at least beneficial, for T-cell lysis of the tumor cells. Therefore, the presence of the murine 3 domain on the tumor may be critical in our model for the recognition and elimination of the HLF16 tumor cell line by T cells. This species-specific 3 requirement is supported by the observation that inefficient interactions between the murine CD8 and the HLA 3 domain limit attempts to restimulate murine anti-HLA specific CTLs in vitro (34, 35, 36)
. The use of stimulator cells and target cells expressing chimeric HLA-A2Kb molecules was found to enhance the generation and detection of HLA class I-restricted CTL responses (37)
. Likewise, we have found that only when splenocytes were stimulated with the H-2Dd-containing HLF16 tumor and the targets contain the chimeric H-2Kb MHC class I did specific lysis occur (data not shown). Furthermore, human JY cells loaded with peptide were not good targets for chromium release by CTLs from HLA-A*0201 transgenic mice (data not shown), stressing the importance of species matching between MHC 3 and CD8 for target cell lysis. Therefore, the success of our in vitro assays was dependent upon stimulator and target cells containing the murine 3 domain.
The results of this study show the value of using human class I transgenic mice to study the variables involved in immunization against HPV. Although numerous vaccination strategies against HPVs have yielded promising results, determining which approaches are most applicable to the clinic proves to be a major challenge in the development of cancer vaccines. This transgenic mouse tumor model may be of great value for assessing the potential of a vaccine before administration to humans. Although the E749–57 epitope always provides full protection in H-2b-dependent tumor models, our HLF16 model showed that vaccination with HPV16 E6- and E7-derived, HLA-A*0201-restricted peptides in IFA was not very effective in the prevention of tumor outgrowth. Therefore, HLA-A*0201 HPV16 peptides would not have been the first choice for a clinical trial based on the results in our HLA-A*0201 tumor model. The disappointing results obtained from HLA-A*0201 HPV16 peptide clinical trials underscore the possible value of HLA transgenic mice to test a vaccine in a model more closely related to humans than regular mice (38 , 39) . If these HPV16 peptides had been first tested in a relevant transgenic model, time and resources could have been directed toward identification and development of more promising vaccination strategies (40) . With the abundance of vaccine candidates against HPV16 (11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 24) , there is a need to distinguish their efficacy. Strategies that appear to be effective in C57BL/6 mice may not lead to a potent response in humans. Therefore, this model offers a preclinical screen for promising vaccines by requiring a human HLA-A*0201 response to induce tumor regression. Several other HLA transgenic mouse strains have been developed that could be used similarly for the assessment of responses restricted by other HLA haplotypes. Because the parental HLF16 tumor is not rejected in naïve mice, this tumor model could also be used to test therapies directed against other tumor antigens by transfecting these antigens into the E6/E7-transformed HLF16 cells, thus allowing for the identification or assessment of HLA-A*0201-specific epitopes against different tumor antigens, such as MAGE, PSA, Her-2/neu or MG50. In conclusion, the transgenic tumor model described here offers an important and useful tool to test the efficacy of vaccination strategies against HPVs and other tumor antigens and may be instrumental for more successful clinical trials.
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FOOTNOTES |
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1 This research was supported by NIH Grants RO1 CA74397 and POI CA97296 (to W. M. K.). G. L. E. is supported by NIH Training Grant T32 AI07508. M. P. V. is a fellow of the Cancer Research Institute. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153.
4 The abbreviations used are: HPV, human papillomavirus; VEE, Venezuelan equine encephalitis; VRP, VEE replicon particle; PSA1, prostate-specific antigen 1; ELISPOT, enzyme-linked immunospot; IFA, incomplete Freund’s adjuvant.
Received 4/11/02. Accepted 8/20/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med., 170: 1091-1101, 1989.3 domain into HLA B27. Nature (Lond.), 348: 642-644, 1990.[Medline]
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) or HLA-A*0201-restricted HPV16 E7 peptides 11–20 and 86–93 in IFA (
). Naïve mice were included to monitor the percentage of tumor take (
). One hundred % of multi-epitope and E7-E6VRP-vaccinated mice remained tumor free, whereas 62% of peptide-vaccinated mice were tumor free. All unvaccinated mice developed a tumor.
