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Clinical Cancer Research Vol. 6, 3406-3416, September 2000
© 2000 American Association for Cancer Research

Advances in Brief

A Phase I Trial of a Human Papillomavirus (HPV) Peptide Vaccine for Women with High-Grade Cervical and Vulvar Intraepithelial Neoplasia Who Are HPV 16 Positive1

Laila Muderspach, Sharon Wilczynski, Linda Roman, Liz Bade, Juan Felix, L. A. Small, W. Martin Kast, Grace Fascio, Verna Marty and Jeffrey Weber2

Divisions of Gynecologic Oncology [L. M., L. R., J. F.], Medical Oncology [L. B., V. M., J. W.], and Pathology [J. F.], University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, California 90033; Cancer Immunology Program, Loyola University School of Medicine Chicago, Illinois 60153 [L. A. S., W. M. K.]; Cardinal Bernardin Cancer Center, Maywood, Illinois 60153 [W. M. K., L. A. S.]; and Department of Pathology, City of Hope Medical Center, Duarte, California 91010 [S. W.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Eighteen women with high-grade cervical or vulvar intraepithelial neoplasia who were positive for human papillomavirus (HPV) 16 and were HLA-A2 positive were treated with escalating doses of a vaccine consisting of a 9-amino acid peptide from amino acids 12–20 encoded by the E7 gene emulsified with incomplete Freund’s adjuvant. Starting with the eleventh patient, an 8-amino acid peptide 86–93 linked to a helper T-cell epitope peptide with a covalently linked lipid tail was added. Patients with colposcopically and biopsy-proven cervical intraepithelial neoplasia/vulvar intraepithelial neoplasia II/III received four immunizations of increasing doses of the vaccine each 3 weeks apart, followed by a repeat colposcopy and definitive removal of dysplastic tissue 3 weeks after the fourth immunization. Patients were skin tested with the E7 12–20 peptide as well as control candida, mumps, and saline prior to and after the series of immunizations. Peripheral blood mononuclear cells were obtained by leucopheresis prior to and after the series of immunizations for analyses of CTL reactivity to the E7 12–20 and 86–93 epitope sequences. The presence of HPV 16 was assessed by DNA PCR on cervical scrapings and the biopsy specimens after vaccination. Pathology specimens were analyzed before and after vaccination for the presence of dysplasia, and the intralesional infiltrate of CD4/CD8 T-cells and dendritic cells was measured by immunohistochemical staining. Only 3 of 18 patients cleared their dysplasia after vaccine, but an increased S100+ dendritic cell infiltrate was observed in 6 of 6 patients tested. Cytokine release and cytolysis assays to measure E7-specific reactivity revealed increases in 10 of 16 patients tested. No positive delayed type hypersensitivity skin test reactivity was shown in any patient to HPV E7 12–20 before or after vaccinations. Virological assays showed that 12 of 18 patients cleared the virus from cervical scrapings by the fourth vaccine injection, but all biopsy samples were still positive by in situ RNA hybridization after vaccination. Six patients had partial colposcopically measured regression of their cervical intraepithelial neoplastic lesions in addition to the three complete responders. The data establish that a HPV-16 peptide vaccine may have important biological and clinical effects and suggest that future refinements of an HPV vaccine strategy to boost antigen-specific immunity should be explored.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
High-grade CIN/VIN3 precede invasive squamous cell carcinoma in most if not all patients with this diagnosis (1 , 2) . Cervical and vulvar cancers usually develop in abnormal squamous epithelium, with a continuum of morphological changes from mildly atypical to anaplastic, frankly malignant cancer cells. The detection of HPV is associated with a 10-fold or greater risk of cervical, vulvar, or vaginal neoplasia compared with a control population of women without HPV (3) . Women with HPV types 16 and 18 were more likely to develop CIN II/III than those with other HPV types. Of the invasive carcinomas, 85–90% of cervical lesions and 35–50% of the vulvar lesions are positive for high-risk HPV types (4 , 5) . Expression of the E6 and E7 early HPV genes appears to link high-risk HPV types to cervical/vulvar neoplasia and invasive carcinoma. Expression of the HPV E6 and E7 genes is necessary and sufficient for transformation of primary keratinocytes and induction of a dysplastic phenotype in human keratinocyte culture (6 , 7) . Ongoing expression of these transforming proteins may be necessary to maintain neoplasia in vitro (8) . HPV E7, a 98-amino acid nuclear protein, binds the retinoblastoma tumor suppressor protein, pRB, as well as a number of other cellular proteins, and serves as a transcriptional activator (9) . The presence of antibodies to E7 correlates with an increased risk for, and worsening stage of, cervical cancer; 20% of women with cervical cancer have measurable E7 antibodies (10) . E7 is expressed in cervical carcinomas, vulvar carcinomas, and their derivative cell lines, as well as in CIN lesions. Because of the consistent detection of E7 in CIN lesions associated with HPV 16, its role in the promotion on oncogenesis, and the recognition that E7 contains class I-restricted T-cell epitopes in several murine and human experimental systems (11 , 12) , it seemed reasonable to attempt to immunize women with high-grade CIN/VIN against E7 as a prevention strategy for cervical/vulvar carcinoma.

CTLs recognize antigens by the binding of their clonotypic TcRs to the processed endogenous peptides associated with class I molecules. Purified epitope peptides derived from different animal viruses have been shown to induce high-affinity CTLs and protect mice against a lethal challenge with infectious viruses (13, 14, 15, 16, 17, 18) . HLA-restricted HIV epitope peptides emulsified in IFA primed a specific murine CTL anti-HIV response (19 , 20) , and immunization with HPV E6 and E7 peptides with IFA induced protection against lethal challenge with E6- and E7-expressing tumor cells in mice (13) . On the basis of this preclinical rationale, we performed a Phase I clinical trial in which two HPV 16 E7 peptides known to be recognized by CTLs were used in escalating doses with IFA to treat patients with high-grade CIN/VIN. In addition to the toxicity and tolerability of the vaccine, immune, virological and clinical response end points were assessed in this clinical trial.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
All patients had unresected and measurable CIN/VIN II/II by the 1991 modified Bethesda classification system and had tissue obtained by a colposcopically directed biopsy. All patients were required to have a lesion that was completely delineated by colposcopy performed 1 month after biopsies were taken or have recurrent positive PAP smears showing squamous intraepithelial lesion II/III. For CIN patients, an endocervical curettage was required to be negative for preinvasive changes or invasive carcinoma. Patients could not have a history of any invasive cancer for at least 5 years. HPV 16 was required to be positive on cervical scrapings by a DNA PCR assay. Eligibility criteria included Eastern Cooperative Oncology Group performance status of 0 or 1, age 18 or greater, creatinine of <1.5 mg/dl, bilirubin of <2.0 mg/dl, platelets of 100,000 per mm3 or more, hemoglobin of 9 g/dl or more, and total WBCs of 3,000 per mm3 or greater. Hepatitis C antibody and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 positive by a microcytotoxicity assay. Patients treated previously for cervical dysplasia were eligible if their therapy was completed at least 90 days prior to entering the protocol. Patients were required to practice an effective form of birth control during the time on trial. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute, and the Los Angeles County and University of Southern California Institutional Review Board. The trial was conducted in accordance with an assurance filed with and approved by the United States Department of Health and Human Services.

Patients were excluded if they had an endocervical curettage indicating preinvasive changes or invasive cancer, previous pelvic irradiation, prior in utero diethylstilbestrol exposure, dependence on steroids, known HIV positivity, or active autoimmune disease (systemic lupus, rheumatoid arthritis, and others).

The HPV-16 E7 12–20 peptide (Ref. 21 ; MLDLQPETT) was produced by Peninsula Labs, Inc. (Belmont, CA). The peptide was provided with Montanide ISA 51 under an Investigational New Drug application held by the Cancer Therapy Evaluation Program of the National Cancer Institute. The final vialed product was produced by the Monoclonal Antibody Production Facility/BioWhittaker. The peptide was produced to Good Laboratory Practice standards. Sterile vials of HPV E7 peptide were stored at 2–8°C and protected from light. E7 12–20 peptide was dissolved in water and filtered through a 0.22 µm Millipore filter. Analysis of the lot to be used in the Phase I study of this protocol demonstrated that there was no pyrogenicity in a rabbit assay and <0.01 endotoxin units/100 µl of endotoxin in a Limulus amebocyte assay. General safety testing in BALB/c mice was satisfactory. Vials containing three concentrations of the peptide were available as 207, 617, and 2057 µg/ml. Each vial contained 1 ml of peptide solution. Peptide was provided by the Cancer Therapy Evaluation Program of the National Cancer Institute (Bethesda, MD) as the trifluroacetate salt in DMSO. The vials of peptide contained no preservative. Montanide ISA-5 1 (IFA) was manufactured by Seppic, Inc. and supplied as glass ampuls containing 3 ml of sterile adjuvant solution without preservative.

An appropriate amount of HPV E7 12–20 was diluted with sterile saline and added in a 1:1 volume to Montanide ISA 51 and then mixed in a Vortex mixer (Fisher, Inc., Alameda, CA) for 10 min at room temperature. The resulting emulsion was injected deeply s.c. in the lateral thigh in a volume of 1 or 2 ml using a glass syringe. Alternating thighs were used for a total of four injections, which were done 3 weeks apart.

Good Manufacturing Practice grade lipopeptide consisting of linker peptide (KSS), the helper peptide PADRE-965.10 (AJXVAAWTLKAAA), and the E7 peptide 86–93 [Ref. 22 ; TLGIVZPI, where aminobutyric acid (Z) is substituted for cysteine (C) at position 91 of the HPV epitope] was produced by Cytel Corp. (San Diego, CA). This 24-amino acid oligomer [(PAM)2 (KSSAKXVAAWTLK-AAA-TLGIVZPI)] was provided under a National Cancer Institute, Cancer Therapy Evaluation Program Investigational New Drug application. Vials contained a solution of HPV-16 E7 86–93 lipopeptide at a concentration of 5 mg/ml in DMSO with 0.1% trifluoracetic acid. Each vial contained 2.0 ml of solution for a total of 10 mg of the lipopeptide per vial. Vials of lipopeptide contained no preservative. The Recombinant Protein Production Facility of the Biological Resources Branch, Biological Response Modified program placed the peptide material into vials. The E7 lipopeptide was diluted with sterile saline and injected deeply s.c. in the lateral thigh in a volume of 1 or 2 ml using a glass syringe. Alternating thighs were used for a total of four injections, which were done 3 weeks apart.

Eighteen patients had a leucopheresis with an exchange of ~5 liters of blood volume performed within 2 weeks before beginning vaccinations and 3 weeks after the final vaccination to collect PBMCs, which were frozen for future analysis. Skin tests were performed using 50 µg of the HPV-16 E7 peptide in aqueous solution injected intradermally in a volume of 100 µl using a tuberculin syringe and a 27-gauge needle. Candida extract and mumps provided a positive control, and saline was a negative control for assessment of delayed-type hypersensitivity. At least 5 mm of induration or erythema above and beyond that shown by saline read 48 h after intradermal injection were required to score a HPV E7 12–20 skin test as positive.

Pheresis samples were processed to purify PBMCs by sedimentation on a Ficoll-Hypaque cushion (Pharmacia, Alameda, CA) and extensive washing in HBSS. Cells were frozen in 40% human AB serum (Gemini Bioproducts, Calabasas, CA), 50% RPMI (Life Technologies, Inc., Grand Island, NY) and 10% DMSO (Sigma Chemical Co., St. Louis, MO) and stored in a liquid nitrogen freezer at -168°C until use.

Cytokine release assays were performed using peptide-stimulated T cells as effector cells. Peptide-stimulated T cells were produced by incubating 2 x 106 thawed PBMCs with 10 µg/ml HPV 16 E7 12–20, 86–93, or FLU-MI in wells of a 24-well plate (Corning, Oneonta, NY). Cells were plated in IMEM with 10% human AB serum. Two days later, IL-2 (kindly provided by Chiron, Emeryville, CA) was added at 50 IU/ml. Fresh IL-2 was added every 3–4 days. After 10, days, the T cells were restimulated with thawed autologous PBMCs pulsed with 10 µg/ml of peptide at 37°C for 2 h and irradiated with 3000R. IL-2 was again added 48 h later at 50 IU/ml. T cells were restimulated with peptide-pulsed PBMCs every 7 days and after 3 restimulations were harvested for immune assays. For the cytokine release assay, 100,000 peptide-stimulated, T cells were harvested at least 5 days after the last restimulation and incubated with 100,000 T2 cells pulsed with 10 µg/ml HPV E7 12–20, 86–93, or FLU M1 peptide or Caski cells as targets in a total volume of 1 ml of RPMI 1640 without serum for 18 h in a 5% CO2 incubator at 37°C. Neither the effectors nor the targets were irradiated. Supernatants were collected, spun briefly at 14,000 x g to pellet cells and debris and frozen at -80°C until assays were done. IFN-{gamma} was detected in supernatants using an antihuman IFN-{gamma} Quantikine ELISA kit (R and D Systems, Minneapolis, MN).

Chromium release assays were performed using the same effectors and targets as in the cytokine release assays, but 5000 targets labeled for 2 h with 51Cr were plated in each well of a 96-well, round-bottomed plate (Corning). Effectors (150,000, 50,000, 15,000, and 5,000) were added to a total volume of 200 µl for final E:T ratios of 30:1, 10:1, 3:1, and 1:1. Twenty-fold excess of K562 cells was added to suppress natural killer activity. E:T mixtures were spun down at 500 x g for 5 min and then incubated for 4 h in a 5% CO2 incubator at 37°C. Supernatants were harvested using a Skatron collecting apparatus, and the liquid-impregnated filters were counted on a Packard gamma counter. The percentage of specific chromium release was measured as:

For TcRs assays, testing for signal transduction molecules was done according to the technique described by Nieland et al. (23) . Assays were scored as the percentage of a battery of 10 normal controls.

Specimens for HPV DNA testing of cervical scrapings were collected in transport media (Digene, Silver Spring, MD) and stored at -20 degrees until processing. HPV typing was performed by PCR as described previously (24, 25, 26) . Briefly, DNA extracted from a cervical scraping was amplified with ß-globin primers to confirm the presence of amplifiable DNA. Consensus primers in the HPV L1 gene MY09/MY11, and type-specific primers for HPV 6, 16, and 18 as well as the reaction conditions have been published. Every reaction set up contained appropriate positive and negative controls. The amplified PCR product was electrophoresed in a 2% agarose gel and stained with ethidium bromide for detection of a visual product. Eligibility criteria for the trial included a positive visual product with the consensus as well as the HPV 16 type primers. All HPV 16 isolates were confirmed by Southern blotting and transferred to nylon membranes (MagnaNT; MSI, Inc.). Products were hybridized overnight with 32P randomly labeled probes for HPV 6, 16, and 18. The membranes were washed four times under stringent conditions with 2x SSC with 0.1% SDS at 48°C and 0.1x SSC with 0.1% SDS at 60°C and exposed to X-ray film with an intensifying screen at -80°C for 4 days.

In situ hybridization for HPV 16 RNA was performed as described previously. After deparaffinization, rehydration, and proteinase K digestion for 30 min at 1 µg/ml (Boehringer Mannheim, Indianapolis, IN), the sections were acetylated in 0.25% acetic acid anhydride and then dehydrated through graded ethanols. HPV riboprobes prepared from pBluescript plasmids were labeled with 35S-labeled UTP and reduced to ~150 bp by alkaline hydrolysis. Sections were hybridized with both sense and antisense strand HPV 16 riboprobes at 45°C in hybridization solution containing 50% formamide, 10% dextran sulfate, 10 mM/l Tris-HCl (pH 7.4), 2x SSC, 1 mM/l EDTA, 500 mg/ml Escherichia coli tRNA, and 1x Denhardt’s solution. After hybridization, the slides were washed in 4x SSC for 20 min, incubated with RNase A (10 µg/ml) at 37°C for 30 min, followed by an additional 30-min wash with 0.1x SSC at 55°C, then dehydrated through graded ethanol containing 300 mM/l ammonium acetate, and coated with photographic emulsion (Kodak, New Haven, CT). Duplicate slides were exposed for 2–4 weeks at 4°C and then developed and lightly counterstained with hematoxylin. The slides were examined under dark-field microscopy, and the signal in the epithelium with the most severe dysplasia were scored 1+ to 3+, with 1+ just above background, 2+ a moderate signal, and 3+ a strong signal with focally very strongly positive individual cells.

Immunohistochemistry for CD3, CD4, CD8, and S100 was performed on formalin-fixed, paraffin-embedded sections according to standard procedures for heat-induced epitope retrieval, as described previously. CD3, CD8, and S100 antibodies (Dako Corp., Carpinteria, CA) were used at dilutions of 1:100, 1:25, and 1:400, respectively, and CD4 (Novacastra Laboratory, Newcastle-on-Tyne, United Kingdom) at a dilution of 1:40. Positive and negative controls included lymph node (CD3 and CD4) and tonsil tissue (CD8), whereas a multi-tissue block of tumor and neural tissue was the S100 control. Antigen-antibody complexes were detected by avidin-biotin technique (Vector Elite kit; Vector, Burlingame, CA) per the manufacturer’s directions with 3',3'-diaminobenzidine as the chromogen.

To estimate the number of intraepithelial DCs, the number of S100-positive nuclei were counted in 3–5 mm of the cervical epithelium with the highest grade of dysplasia and then averaged per mm. Most of the biopsy specimens had insufficient adjacent uninvolved epithelium to evaluate. In the cone biopsies taken after treatment the number of S100-positive DCs, nuclei were counted in a region of the cone that had histologically normal epithelium and little inflammation.

The number of T cells positive in the superficial stroma that were immunoreactive with CD3, CD4, and CD8 were scored 1+ to 4+ based on the following scale: 1+ showed a few scattered positive cells; 2+, occasional clusters or patchy groups of positive cells; 3+, diffuse infiltrate; and 4+, a heavy infiltrate.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Demographics and Toxicities in Vaccinated Patients.
A total of 18 patients were treated on the HPV 16 E7 12–20/86–93 peptide trial. Three cohorts of four patients each received increasing doses of the E7 12–20 peptide with IFA, and subsequent patients received 2000 µg of the E7 12–20 peptide with IFA, together with the E7 86–93 lipopeptide at doses of 1 or 10 mg. The demographic data on the first 18 patients are shown in Table 1Citation . The median age was 29, with 16 CIN II/III and 2 VIN II/III patients. All patients were HLA-A2 positive, and all patients had a Virapap showing HPV type 16 by DNA PCR analysis. All patients but one had a measurable lesion in the cervix or vulva that was biopsy proven at least 1 month prior to therapy and was colposcopy positive 1 month after initial biopsy to rule out spontaneous regression; one patient had a history of carcinoma in situ of the cervix with positive PAP smears postoperatively but no lesions positive by biopsy. All patients gave informed consent and had a pretherapy pheresis within 2 weeks prior to starting vaccinations and within 4 weeks after the fourth and last vaccine injection.


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  		Table 1 Demographics and clinical characteristics

 
The toxicity of the vaccine preparations was modest, with 17 of 18 patients demonstrating grade I or II local reactions and/or granuloma formation, with erythema, edema, and warmth persisting for 3 weeks after injections at all doses. Grade II lethargy, weakness, nausea, diarrhea, and fever occurred in patients at the highest to the lowest doses of 12–20 E7 peptide, suggesting that systemic symptoms were not dose related. The only patients with persistent granulomas received the larger volume of E7 12–20 peptide at 2000 µg/dose, requiring a 2-ml injection of IFA/peptide that was mixed 1:1. No grade III/IV toxicity was observed, and no doses of vaccine were withheld because of toxicity. Toxicities of the E7 12–20 peptide/IFA and 86–93 lipopeptide vaccine are shown in Table 2Citation below.


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  		Table 2 Toxicities of HPV peptides

 
Virological Results in Vaccinated Patients.
Of the 18 patients in whom full PCR data are available, clearing of virus by the time of the definitive procedure took place in 12, a decreased signal was detected in 3, and no change in the intensity of the Pap-derived DNA PCR signal was seen in 3. Table 3Citation shows the evolution of viral PCR signals for 18 patients. In the published literature, little information is available about Virapap detection of HPV by PCR after a LEEP, but we examined Virapaps over time after LEEP for HPV 16 by DNA PCR in a sample of 16 patients who were HLA-A2 negative or had an unsatisfactory colposcopy and could not go on the vaccine study. In 16 of 16 cases, virus was positive prior to LEEP but cleared within 4 months afterward from the mucosae (data not shown). Of note is that for six patients who had adequate biopsy tissue with viable dysplastic tissue to study after vaccination, an in situ RNA hybridization signal was detected in tissue removed from all LEEP or excision specimens of these patients at the time of their definitive procedure. Interestingly, 3 of 6 samples (patients 3, 8, and 15) had a much weaker signal after vaccination (data not shown). The PCR data from immunized patients in this study suggest that in a significant proportion of cases (15 of 18), a HPV 16 viral DNA signal derived from swab material of the cervix or vulva disappears or diminishes after peptide vaccine therapy and prior to definitive surgery, but that a RNA signal in the dysplastic tissue itself continues to be detected.


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  		Table 3 Detection of HPV 16 by PCR in patients receiving HPV 16 peptide vaccine

 
Immunological Results in Vaccinated Patients.
All patients in the trial had a minimum 5-liter leucopheresis no more than 2 weeks prior to initiating the vaccine trial and within 4 weeks of the fourth or last vaccine. PBMCs were pulsed with HPV 16 E7 12–20, 86–93, or control FLU M1 matrix peptides and restimulated three times in the presence of low doses of IL-2 (50 IU/ml) as described in "Materials and Methods." Resulting effector cells were tested in a IFN-{gamma} cytokine release assay or a chromium release assay using antigen-specific and nonspecific targets to test whether there was an increase in HPV-specific CTL activity detected in the peripheral blood after vaccinations compared with before. The data shown below in Fig. 1Citation for the first 16 patients represent cross-specificity assays to detect antigen-specific T cells that release cytokine (IFN-{gamma}) before and after immunizations. Total PBMCs were thawed simultaneously and split into two parts. One aliquot before and after vaccination was repeatedly stimulated with the FLU M1 matrix 58–66, A2-restricted epitope peptide, and the other with HPV E7 12–20 peptide. After three weekly restimulations, the resulting effector cells were incubated with HLA-A2+ targets expressing the E7 12–20, FLU 58–66, or no peptide. After 18 h, supernatants were collected, frozen, and later thawed and assayed for IFN-{gamma} by ELISA. For 10 of 16 patients tested, there is a clear augmentation of HPV E7-specific IFN-{gamma} release after vaccination, without a significant change in the FLU-specific reactivity (FLU data not shown). Positive assays were seen for patients 1, 4, 5, 7, 8, 10, 11, 13, 15, and 16. FLU-stimulated cells revealed no E7-specific reactivity relative to background, nor did E7specific effectors show reactivity against FLU, demonstrating appropriate cross-specificity and suggesting that there is a significant augmentation of E7-specific T-cell immunity as a result of vaccination. There does not appear to be a dose response, with high levels of cytokine release at all doses of peptide used, although the numbers are obviously quite small. Patients 11–16 received the 86–93 lipopeptide, and they had separate aliquots of PBMCs stimulated with the 86–93 peptide, with no responders observed in cytokine release assays (data not shown). All samples were used for a repeated cytokine release assay to verify the results of the assay shown in Fig. 1Citation , and the results were reproducible (data not shown).



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  		Fig. 1. Immune response to HPV-E7 peptide vaccine shown by increased cytokine release. Effector cells were prepared as described in "Materials and Methods," and 100,000 effector cells were restimulated with HPV E7 12–20 or FLU M1 peptide-pulsed PBMCs after the third restimulation in vitro were plated with 100,000 peptide-pulsed T2 target cells or unpulsed T2 cells in a 24-well plate in complete medium for 18 h in a volume of 1 ml. The supernatant was harvested and then spun in a microcentrifuge at 14,000 x g for 30 s to pellet cells and debris. Supernatants were removed and used to measure IFN-{gamma} release using a commercial ELISA kit as described in "Materials and Methods." Graphically shown are the means of duplicates for values obtained with T2 cells pulsed with E7 12–20 peptide at 10 µg/ml, with the actual values for cytokine release listed above each column. Figures for FLU-stimulated cells are not shown but were positive for 16 of 18 patients at >100 pg/ml both before and after vaccination. IFN-{gamma} was measured in pg/ml. Values indicate E7 12–20-specific cytokine release, with >100 pg/ml cytokine released after vaccination compared with baseline scored as positive. Similar results were obtained with repeated experiments for all patients.

 
Chromium release assays were also performed to verify that cells releasing cytokine were also lytic. The data in Fig. 2Citation suggest that there is a correlation between the cytokine and chromium release assays, because 10 patients (nos. 1, 4, 5, 7, 8, 10, 11, 13, 15, and 16) exhibited an immune response with increased cytokine secretion after HPV 16 E7 12–20 stimulation, and 8 of those 10 samples showed augmented E7 12–20-specific cytolysis in Fig. 2Citation . For the six patients analyzed who also received the 86–93 lipopeptide, augmented chromium release was observed in three, shown for patient nos. 11, 13, and 16 in Fig. 3Citation . The above data suggest that reactive T cells detected by cytokine release assays after E7 peptide vaccination are also lytic for E7-expressing targets.



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  		Fig. 2. Immune response to HPV-E7 vaccine shown by increased cytolysis. Effector cells were prepared as described in "Materials and Methods" after three restimulations in vitro and plated at varying E:T ratios in 96-well plates for 4 h with 5000 chromated T2 target cells pulsed or not pulsed with the HPV E7 12–20 or E7 86–93 epitope peptides. Patient identifying numbers and cohort are shown on the abscissa, and the values for chromium release before and after vaccination for E7 12–20- and E7 86–93-pulsed T2 cells as targets at E:T ratios of 30:1 and 10:1 with HPV E7 12–20- and E7 86–93-stimulated effector cells, respectively, are shown on the ordinates of Figs. 2Citation 3Citation . All values shown are the means of triplicates with spontaneous and maximum release values calculated as indicated in "Materials and Methods." Similar results were obtained when the experiment was repeated.

 


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  		Fig. 3. Immune response to HPV-E7 vaccine shown by increased cytolysis. Effector cells were prepared as described in "Materials and Methods" after three restimulations in vitro and plated at varying E:T ratios in 96-well plates for 4 h with 5000 chromated T2 target cells pulsed or not pulsed with the HPV E7 12–20 or E7 86–93 epitope peptides. Patient identifying numbers and cohort are shown on the abscissa, and the values for chromium release before and after vaccination for E7 12–20- and E7 86–93-pulsed T2 cells as targets at E:T ratios of 30:1 and 10:1 with HPV E7 12–20- and E7 86–93-stimulated effector cells, respectively, are shown on the ordinates of Figs. 2Citation 3Citation . All values shown are the means of triplicates with spontaneous and maximum release values calculated as indicated in "Materials and Methods." Similar results were obtained when the experiment was repeated.

 
Pathological Results in Vaccinated Patients.
No difference was observed in the numbers of immunoreactive CD4 and CD8 cells in the limited number of specimens for which viable dysplastic tissue was measured both before and after vaccination. However, the mean number of intraepithelial S100-positive DCs in dysplastic cervical epithelium increased by >2-fold in biopsies after vaccination (18.1 versus 8.1), as shown in Table 4Citation . In the cone biopsies after vaccination, the adjacent nondysplastic squamous epithelium had fewer DCs than did the cervical intraepithelial neoplasia. Fig. 4Citation shows an example of a CIN III lesion before vaccination with few infiltrating DCs by immunohistochemical staining in the left panel and a large increase in the DC infiltrate in the postvaccination LEEP specimen in the right panel. Decreases in Langerhans cells, which are antigen-presenting DCs present in epithelial tissues, are reported in HPV-infected cervical epithelium, and there is some evidence to suggest that fewer Langerhans cells can be found in cervical lesions that persist compared with lesions that regress (27 , 28) . The current data suggest that in a modest number of carefully prepared samples at different doses of the HPV peptide vaccine, significant increases in DC infiltration were seen after vaccination.


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  		Table 4 Immunohistochemical detection of S100-positive dendritic cells

 


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  		Fig. 4. A, cervical biopsy showing CIN 3 from patient prior to vaccine treatment and immunohistochemically stained for S100 protein. The dysplastic cervical epithelium had 10.5 S100-positive cells/mm. x200. B, dysplastic cervical epithelium from the same patient’s cone biopsy after vaccination and immunohistochemically stained for S100 protein. There were 23.4 S100-positive dendritic cells/mm of dysplastic epithelium. x200.

 
TcR Signal Transduction Defects in CIN/VIN Patients.
Frozen PBMCs were used to assess levels of TcR {zeta} chain signal transduction molecules, which have been found to be deficient in cancer patients in general and women with cervical cancer in particular (23 , 29) . T cells that have deficient levels of TcR transduction molecules are unlikely to be fully functional in vivo or in vitro. Of the first 16 women in the trial, all except 2 had significant defects in TcR {zeta} by intracellular flow cytometry staining (mean TcR, 69.3 ± 4.5% before vaccination and 74.1 ± 11.2% after vaccination). Normal levels were established using a large number (>10) of normal healthy controls, and the values for the CIN/VIN patients are expressed as a percentage of normal controls. No significant changes in TcR {zeta} were seen after vaccination. These data emphasize that women without invasive malignancy have clear evidence of immunosuppression, suggesting that strategies to boost immune responses and immune competence in vaccinated women with CIN/VIN are important.

Clinical Results in Vaccinated Patients.
A total of 9 of 17 evaluable patients had partial or complete regression of their CIN lesions. Three had complete regression, and 6 had >50% shrinkage of their colposcopically measured disease, as summarized in Table 5Citation . No clear correlation was observed between immune responses and clinical regression or disappearance of CIN. Patients at all doses of vaccine had regression of disease, indicating that a dose response was unlikely.


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  		Table 5 Clinical effects of HPV peptide vaccine

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The detection of HPV is associated with a significant risk of cervical or lower genital tract neoplasia and is regarded as a predisposing factor in the development of invasive cancer compared with a control population of women without HPV (3) . In a study of 235 women treated in a sexually transmitted disease clinic who were HPV positive but had negative cervical cytology, there was an 11-fold risk of developing CIN II/III within a 2-year follow-up period, compared with a control population with similar sexual histories but negative for HPV (30) . Women with HPV types 16 and 18 were more likely to develop CIN II/III than those with other HPV types. The HPV types have been divided into groups at low, high, and intermediate risk for the development of intraepithelial neoplasia and cancer. In most studies, ~75% of high-grade cervical intraepithelial lesions are positive for high- or intermediate-risk HPV types, such as 16 and 18, compared with 40–50% of vaginal and 80–90% of vulvar epithelial lesions. The spectrum of HPV-associated clinical findings in women ranges from genital warts to squamous intraepithelial neoplasia and invasive carcinoma (31) . HPV types 6 and 11 are associated with genital warts (condyloma accuminata; Ref. 32 ) and types 16 and 18 with intraepithelial and invasive lesions. HPV 16 is the most common type found in squamous carcinomas (33) , and HPV 18 is most common in adenocarcinomas and small cell neuroendocrine cervical carcinomas (34 , 35) . These data provide a strong justification for devising immunization strategies against high-risk HPV types to prevent progression from low-grade CIN/VIN to high-grade disease, recurrence of high-grade dysplasia, and the occurrence of invasive cervical/vulvar cancer.

HPV-specific serological, T helper, and T cytolytic immune responses have been demonstrated in patients with high-grade CIN/VIN and cervical cancer. Seropositivity directed against the E1, E2, E4, and E7 proteins in virus-like particles has been documented in patients positive for HPV 16 by PCR DNA (36, 37, 38) . T-helper responses to the L1, E2, E4, and E7 proteins can be detected by proliferation of peripheral blood mononuclear cells in patients with high grade CIN/VIN (38, 39, 40, 41, 42, 43, 44, 45) . Proliferative T-cell responses correlate with high-grade lesions and HPV 16 DNA positivity by PCR (40 , 44 , 45) . In mice, experiments to detect E7 CTL reactivity have been performed using murine tumor cells transfected with the full-length HPV 16 genome or E7 cDNA. Specific CTL reactivity has been generated in mice immunized with tumor cells that express E7 and the T-cell costimulatory molecule B7 (46) or infected with a vaccinia virus construct containing the full-length E7 cDNA (47) . A murine H-2-restricted E7 epitope peptide has been used to immunize mice in combination with IFA. Immunized mice developed protection against a subsequent challenge with a lethal dose of E7-expressing tumor cells (13) . Specific anti-E7 CTLs were generated using peptide vaccination with IFA or after peptides were pulsed onto autologous murine splenocyte-derived DCs (48 , 49) . The use of a lipid-tailed peptide construct resulted in increased CTL induction in mice compared with the peptide with adjuvant (50) . In mice, a T-cell line that recognizes that epitope eradicated established HPV-16-induced tumors in mice.

Nine- and 10-amino acid peptides from HPV-16 E7 were defined by strong binding to HLA-A2, and the immunogenicity of a number of A2-binding peptides was tested in vivo in HLA-A2 transgenic mice. Three peptides were defined that were immunogenic both in transgenic mice and in CTL induction experiments using PBMCs from HLA-A2 healthy donors derived from the 11–20, 82–90, and 86–93 amino acid sequences (51) .

E7-specific CTL cells have been generated from the peripheral blood and lymph nodal tissue of HPV-16-positive women with cervical dysplasia and cervical cancer by in vitro restimulation with autologous antigen-presenting cells pulsed with HPV-16 E7 peptides (52 , 53) . CTL responses against HPV-16 E7 appeared to be more potent in women who were virus positive but CIN negative than in those who were CIN positive, suggesting a role for specific CTL responses in tumor progression (54) . Autologous peripheral blood cells and DCs pulsed with E7 peptides or protein were potent inducers of anti-HPV immunity in cervical cancer patients. (55, 56, 57) . When normals without HPV infection were tested, no E7-specific CTLs could be detected. Women with stage IV cervical cancer were immunized with a lipopeptide construct identical to that used in this trial, and only six patients mounted a weak immune response against the E7 86–93 peptide sequence, without evidence of clinical benefit (58, 59, 60) . Vaccination with CTL epitope peptide, strong adjuvant, and nonspecific help might prime CTLs against the weakly immunogenic epitope and result in clearance of HPV. It would seem that women with a lower disease burden and preinvasive disease are more logical candidates for an antigen-specific immunotherapy than women with bulky invasive disease, prior chemotherapy, poor performance status, and profound immunosuppression.

The data presented herein suggest that a vaccine consisting of a HPV 16 E7 peptide administered with IFA added to a HPV E7 lipopeptide stimulates an immune response in a significant proportion of HLA-A2-positive patients with CIN/VIN II/III evidenced by cytokine or chromium release assays. The peptide vaccine was well tolerated, with side effects mostly consisting of local pain and granuloma formation. No grade III or IV toxicity occurred in this trial of 18 women. All 17 patients with CIN had a definitive excision procedure after their series of four vaccines was finished, and only 3 of 17 evaluable patients had complete regression of their lesion pathologically, although an additional 6 had partial regression of their CIN lesion. These data must be interpreted with the acknowledgment of a reported 20–30% rate of spontaneous regression noted in patients with high-grade dysplasia (61) , and although disappearance of CIN in 3 patients and partial regression in 6 is encouraging, only the performance of a randomized trial with a placebo control group will permit definitive conclusions on the efficacy of this vaccine regimen. In six pre- and postvaccine specimens examined by microscopy, no change in CD8+ or CD4+ T-cell infiltrate was seen on immunohistochemical staining, but significant and consistent increases in infiltrating S100-positive DCs were observed, suggesting that vaccination resulted in infiltration of the dysplastic lesion with antigen-presenting DCs. These results should be interpreted in light of reports that Langerhan’s cells are decreased in dysplastic cervical epithelium, and fewer Langerhans cells are found in cervical lesions that persist compared with lesions that regress (62) . Because infiltration of tumors with DCs may correlate with a positive outcome, augmented infiltration of dysplastic tissue with DCs after vaccination may represent a beneficial surrogate marker. A possible mechanism to explain the augmented DC infiltrate might be increased expression of chemokines that are known to impact on the migration of DCs, such as MIP-3{alpha} or MIP-3ß, by inflammatory cells that infiltrated a regressing lesion after vaccination (63) .

In 15 of 18 patients, disappearance or decreased intensity of a HPV DNA PCR signal was detected prior to and at the time of LEEP, indicating that in a sensitive assay, the majority of patients had clearance of HPV 16 from the cervical and/or vulvar tissue after vaccination with peptides. All biopsy specimens of dysplastic tissue tested had evidence of HPV 16 mRNA transcription by in situ hybridization at the time of definitive removal, indicating that virus genetic material was present and had not been cleared from the dysplasia.

The significant findings of this Phase I study were that the majority of patients had a detectable immune response in peripheral blood cells after four injections of the peptide E7 12–20 vaccine. Immune responses in 10 of 16 patients by cytokine release assay was confirmed by cytolysis assays in 8 of the 10, suggesting that cytokine-secreting and CTLs were augmented in the peripheral blood after the HPV 16 E7 12–20 peptide vaccine with IFA. All patients except 2 had a positive FLU-specific cytokine response both before and after vaccination as a positive control for the assay. One of the patients (no. 2) that did not respond to FLU M1 or to E7 12–20 was HLA-A2 subtyped by PCR, revealing the A 0206 subtype,4 which has been shown to bind A0201-associated peptides such as those used in this trial at a greatly reduced level (64) . The augmented immune responses seen in this trial are similar to those observed in a peptide trial in cervical cancer (58) , but these data are the first that we know of demonstrating that patients with high-grade cervical/vulvar dysplasia can be vaccinated against HPV 16 and mount a detectable immune response in the peripheral blood. The data on TcR transduction molecules described in the text are consistent with globally depressed immunity similar to that seen in patients with frank, invasive cancer. The demonstration of decreased levels of class I molecules on dysplastic and frankly neoplastic cervical cells (65 , 66) and the reduction in TcR {zeta} chain detected in our patients with preneoplastic dysplasia suggest that strategies to overcome host immunosuppression will be an important aspect of any effort to prevent cancer by vaccination of high-risk patients.

Because it is likely that continued expression of E7 is required for proliferation and is necessary but not sufficient for malignant transformation, clearance of virus and/or dysplastic cells expressing E7 might induce regression of CIN/VIN II/III, generate long-lasting immunity against HPV 16, and prevent the premalignant changes associated with HPV 16. Long-lasting protection against cervical/vulvar intraepithelial neoplasia would have a significant impact on the incidence of cervical and vulvar carcinoma. The encouraging initial results from the trial described herein indicate that potent, long-lasting levels of T-cell-mediated immune responses might be beneficial to patients with high-grade dysplasia. In future trials, we will continue efforts to prevent cervical/vulvar cancer by augmenting T-cell immunity to a specific, well-characterized tumor antigen using DNA plasmids and heat shock proteins to deliver the immunogen.


   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 Grants RO1-CA67872, RO1-CA74397, RO1-CA/AI78399, and 5P30-CA14089 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at University of Southern California/Norris Comprehensive Cancer Center, Room 6428, 1441 Eastlake Avenue, Los Angeles, CA 90033. Phone: (323) 865-3919; Fax: (323) 865-0061; E-mail: jweber{at}hsc.usc.edu Back

3 The abbreviations used are: CIN, cervical intraepithelial neoplasia; VIN, vulvar intraepithelial neoplasia; HPV, human papillomavirus; PBMC, peripheral blood mononuclear cell; HLA, human leukocyte antigen; HIV, human immunodeficiency virus; IFA, incomplete Freund’s adjuvant; TcR, T-cell receptor; LEEP, loop electrocautery excision procedure; DC, dendritic cell; IL, interleukin. Back

4 F. Marincola, personal communication. Back

Received 2/29/00; revised 5/30/00; accepted 5/31/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Richart R. M. Natural history of cervical intraepithelial neoplasia. Clin. Obstet. Gynecol., 10: 748-758, 1968.
  2. Hall J. E., Walton L. Dysplasia of the cervix: a prospective study of 206 cases. Am. J. Obstet. Gynecol., 100: 662-670, 1968.[Medline]
  3. Schiffman M. H. Recent progress in defining the epidemiology of human papillomavirus infection and cervical neoplasia. J. Natl. Cancer Inst., 84: 394-398, 1992.[Free Full Text]
  4. Lorincz A. T., Reid R., Jenson A. B., Greenberg M. D., Lancaster W., Kurman R. J. Human papillomavirus infection of the cervix: relative risk associations of 15 common anogenital types. Obstet. Gynecol., 79: 328-337, 1992.[Medline]
  5. DeBritton R. C., Hildesheim A., De Lao S. L., Brinton L. A., Sathya P., Reeves W. C. Human papillomaviruses and other influences on survival from cervical cancer in Panama. Obstet. Gynecol., 81: 19-24, 1993.[Abstract/Free Full Text]
  6. Munger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol., 63: 4417-4424, 1989.[Abstract/Free Full Text]
  7. Hudson J. B., Bedell M. A., McCance D. J., Laiminis L. A. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J. Virol., 64: 519-526, 1990.[Abstract/Free Full Text]
  8. Crook T., Morganstern J. P., Crawford L. Continued expression of HPV 16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV 16 plus EJ-ras. EMBO J., 8: 513-520, 1989.[Medline]
  9. Munger K., Werness B. A., Dyson N., Phelps W. C., Harlow E., Howley P. M. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J., 8: 4099-4105, 1989.[Medline]
  10. Galloway D. A. Serologic assays for the detection of HPV antibodies Munoz N. Bosch F. X. Shah K. V. eds. . The Epidemiology of Cervical Cancer and Human Papillomaviruses, : 147-162, IARC Scientific Publications Lyon, France 1992.
  11. Chen L. P., Thomas E. K., Hu S. L., Hellstrom I., Hellstrom K. E. Human papillomavirus type 16 nucleoprotein E7 is a tumor rejection antigen. Proc. Natl. Acad. Sci. USA, 88: 110-114, 1991.[Abstract/Free Full Text]
  12. Chen L., Ashe S., Brady W. A., Hellstrom I., Hellstrom K. E., Ledbetter J. A., McGowan P., Linsley P. S. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA4. Cell, 71: 1093-1102, 1992.[CrossRef][Medline]
  13. Feltkamp M. C., Smits H. L., Vierboom M. P., Minnaar R. P., de Jongh B. M., Drijfhout J. W., ter Schegget J., Melief C. J., Kast W. M. Vaccination with cytolytic T lymphocyte epitope containing peptide protects against a tumor induced by human papillomavirus type 16 transformed cells. Eur. J. Immunol., 23: 2242-2249, 1993.[Medline]
  14. Kast W. M., Roux L., Curren J., Blom H. J., Voordouw A. C., Meloen R. H., Kolakofsky D., Melief C. J. Protection against lethal Sendai virus infection by in vivo priming of virus specific cytotoxic T lymphocytes with a free synthetic peptide. Proc. Natl. Acad. Sci. USA, 88: 2283-2287, 1991.[Abstract/Free Full Text]
  15. Deres K., Schild H., Wiesmuller K. H., Jung G., Rammensee H. G. In vivo priming of virus specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature (Lond.), 342: 561-563, 1989.[CrossRef][Medline]
  16. Gao X-M., Zheng B., Liew F. Y., Brett S., Tite J. Priming of influenza virus-specific cytotoxic T lymphocytes in vivo by short synthetic peptides. J. Immunol., 147: 3268-3272, 1991.[Abstract]
  17. Aichile P., Hengartner H., Zinkernagel R. M., Schultz M. Antiviral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide. J. Exp. Med., 171: 1815-1820, 1990.[Abstract/Free Full Text]
  18. Harty J. T., Bevan M. J. CD8+ cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo.. J. Exp. Med., 175: 1531-1538, 1992.[Abstract/Free Full Text]
  19. Takahashi H., Cohen J., Hosmalin A., Cease K. B., Houghten R., Cornette J. L., DeLisi C., Moss B., Germain R. N., Berzofsky J. A. An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA, 85: 3105-3109, 1988.[Abstract/Free Full Text]
  20. Hart M. K., Weinhold K. J., Scearce R. M., Washburn E. M., Clark C. A., Palker T. J., Haynes B. F. Priming of anti-human immunodeficiency virus (HIV) CD8+ cytotoxic T cells in vivo by carrier free HIV synthetic peptides. Proc. Natl. Acad. Sci. USA, 88: 9448-9452, 1991.[Abstract/Free Full Text]
  21. Nijman H. W., Houbiers J. G., van der Burg S. H., Vierboom M. P., Kenemans P., Kast W. M., Melief C. J. Characterization of cytotoxic T lymphocyte epitopes of a self-protein, p53, and a non-self-protein, influenza matrix: relationship between major histocompatibility complex peptide binding affinity and immune responsiveness to peptides. J. Immunother., 14: 121-126, 1993.
  22. Kast W. M., Brandt R. M., Sidney J., Drijfhout J. W., Kubo R. T., Grey H. M., Melief C. J., Sette A. Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins. J. Immunol., 152: 3904-3912, 1994.[Abstract]
  23. Nieland J. D., Loviscek K., Kono K., Albain K. S., McCall A. R., Potkul R. K., Fisher S. G., Velders M. P., Petersson M., Kiessling R., Kast W. M. PBLs of early breast carcinoma patients of a high nuclear grade tumor unlike PBLs of cervical carcinoma patients do not show a decreased TcR {zeta} expression but are functionally impaired. J. Immunother., 21: 317-322, 1998.
  24. Monk B. J., Cook N., Ahn C., Wilczynski S. P. Comparison of the polymerase chain reaction and Southern blot analysis in detecting and typing human papilloma virus deoxyribonucleic acid in tumors of the lower female genital tract. Diag. Mol. Pathol., 3: 283-291, 1994.[Medline]
  25. Meyer T., Arndt R., Stockfleth E., Flammann H. T., Wolf H., Reischl U. Strategy for typing human papillomaviruses by RFLP analysis of PCR products and subsequent hybridization with a generic probe. Biotechniques, 19: 632-639, 1995.[Medline]
  26. Wilczynski S. P., Oft M., Cook N., Liao S. Y., Iftner T. Human papillomavirus type 6 in squamous cell carcinoma of the bladder and cervix. Hum. Pathol., 24: 96-102, 1993.[CrossRef][Medline]
  27. Fukuda K., Hacisuga T., Nakamura S. Local immune response in persistent cervical dysplasia. Obstet. Gynecol., 82: 941-945, 1993.[Abstract/Free Full Text]
  28. Morelli A. E., Sananes C., Di Paola G., Paredes A., Fainboim L. Relationship between types of human papillomavirus and Langerhans’ cells in cervical condyloma and intraepithelial neoplasia. Am. J. Clin. Pathol., 99: 200-206, 1993.[Medline]
  29. Kono K., Ressing M. E., Brandt R. M., Melief C. J., Potkul R. K., Andersson B., Petersson M., Kast W. M., Kiessling R. Decreased expression of signal transduction {zeta} chain in peripheral T cells and natural killer cells in patients with cervical cancer. Clin. Cancer Res., 2: 1825-1828, 1996.[Abstract]
  30. Koutsky L. A., Holmes K. K., Critchlow C. W., Stevens C. E., Paavonen J., Beckmann A. M., DeRouen T. A., Galloway D. A., Vernon D. Kiviat NBA cohort study of the risk of cervical intraepithelial neoplasia grade II or III in relation to papillomavirus infection. N. Engl. J. Med., 327: 1272-1278, 1992.[Abstract]
  31. Fu Y. S., Huang I., Beaudenon S., Ionesco M., Barrasso R., de Brux J., Orth G. Correlative study of HPV, DNA, histopathology and morphometry in cervical condyloma and intraepithelial neoplasia. Int. J. Gynecol. Pathol., 7: 297-303, 1988.[Medline]
  32. Wagner D., Ikenberg H., Boehm N., Gissmann L. Identification of human papillomavirus in cervical swabs by deoxyribonucleic acid in situ hybridization. Obstet. Gynecol., 64: 767-771, 1984.[Abstract/Free Full Text]
  33. Wilczynski S. P., Bergen S., Walker J., Liao S. Y., Pearlman L. F. Human papillomaviruses and cervical carcinomas: histologic features associated with different viral types. Hum. Pathol., 19: 697-704, 1998.[CrossRef]
  34. Wilczynski S. P., Walker J., Liao S. Y., Bergen S., Berman M. Adenocarcinoma of the cervix associated with human papillomaviruses, Cancer (Phila. ), 62: 1332-1336, 1988.
  35. Stoler M. H., Mills S. E., Gersell D. J., Walker A. J. Small cell neuroendocrine carcinoma of the cervix. A human papillomavirus type 18 associated cancer. Am. J. Surg. Pathol., 15: 28-32, 1991.[Medline]
  36. Muller M., Viscidi R. P., Ulken V., Bavinck J. N., Hill P. M., Fisher S. G., Reid R., Munoz N., Schneider A., Shah K. V. Antibodies to E4, E6 and E7 proteins of human papillomavirus (HPV) type 16 in patients with HPV associated diseases and the normal population. J. Investig. Dermatol., 104: 138-141, 1995.[CrossRef][Medline]
  37. Wideroff L., Schiffman M. H., Hoover R., Tarone R. E., Nonnenmacher B., Hubbert N., Kirnbauer R., Greer C. E., Lorincz A. T., Manos M. M., Glass A. G., Scott D. R., Sherman M. E., Buckland J., Lowy D., Schiller J. Evaluation of seroreactivity to human papillomavirus type 16 virus-like particles in an incident case-control study of cervical neoplasia. J. Infect. Dis., 174: 237-239, 1996.[Medline]
  38. Nonnenmacher B., Hubbert N. L., Kirnbauer R., Shah K. V., Munoz N., Bosch F. X., de Sanjose S., Viscidi R., Lowy D. R., Schiller J. T. Serologic responses to human papillomavirus type 16 (HPV 16) virus-like particles in HPV-16 DNA positive invasive cervical cancer and cervical intraepithelial neoplasia grade III patients and controls from Colombia and Spain. J. Infect. Dis., 172: 19-24, 1995.[Medline]
  39. Nakagawa M., Stites D. P., Farhat S., Judd A., Moscicki A. B., Canchola A. J., Hilton J. F., Palefsky J. M. T-cell proliferative responses to human papillomavirus type 16 peptides: relationship to cervical intraepithelial neoplasia. Clin. Diagn. Lab. Immunol., 3: 205-210, 1996.[Abstract]
  40. de Gruijl T. D., Bontkes H. J., Stukart M. J., Walboomers J. M., Remmink A. J., Verheijen R. H., Helmerhorst T. J., Meijer C. J., Scheper R. J. T-cell proliferative responses against human papillomavirus type 16 E7 oncoprotein are most prominent in cervical intraepithelial neoplasia patients with a persistent viral infection. J. Gen. Virol., 77: 2183-2191, 1996.[Abstract/Free Full Text]
  41. Luxton J. C., Rowe A. J., Cridland J. C., Coletart T., Wilson P., Shepherd P. S. Proliferative T-cell responses to the human papillomavirus type 16 E7 protein in women with cervical dysplasia and cervical carcinoma and healthy women. J. Gen. Virol., 77: 1585-1593, 1996.[Abstract/Free Full Text]
  42. Shepherd P. S., Rowe A. J., Cridland J. C., Coletart T., Wilson P., Luxton J. C. Proliferative T-cell responses to human papillomavirus type 16 L1 peptides in women with cervical dysplasia. J. Gen. Virol., 77: 593-602, 1996.[Abstract/Free Full Text]
  43. Lehtinen M., Hibma M. H., Stellato G., Kuoppala T., Paavonen J. Human T helper cell epitopes overlap B cell and cytotoxic T cell epitopes in the E2 protein of human papillomavirus type 16. Biochem. Biophys. Res. Commun., 209: 541-546, 1995.[CrossRef][Medline]
  44. Kadish A. S., Ho G. Y., Burk R. D., Wang Y., Romney S. L., Ledwidge R., Angeletti R. H. Lymphoproliferative responses to human papillomavirus type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia. J. Natl. Cancer Inst., 89: 1285-1293, 1997.[Abstract/Free Full Text]
  45. de Gruijl T. D., Bontkes H. J., Walboomers J. M., Stukart M. J., Doekhie F. S., Remmink A. J., Helmerhorst T. J., Verheijen R. H., Duggan-Keen M. F., Stern P. L., Meijer C. J., Scheper R. J. Differential T-helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study. Cancer Res., 58: 1700-1706, 1998.[Abstract/Free Full Text]
  46. Lipford G. B., Bauer S., Wagner H., Heeg K. Peptide engineering allows cytotoxic T-cell vaccination against human papillomavirus tumor antigen E6. Immunology, 84: 298-303, 1995.[Medline]
  47. Sarkar A. K., Tortolero-Luna G., Nehete P. N., Arlinghaus R. B., Mitchell M. F., Sastry K. J. Studies on in vivo induction of cytotoxic T lymphocyte responses by synthetic peptides from E6 and E7 oncoproteins of human papillomavirus type 16. Viral Immunol., 8: 165-174, 1995.[Medline]
  48. Ossevoort M. A., Feltkamp M. C., van Veen K. J., Melief C. J., Kast W. M. Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope-based peptide vaccine in protection against a human papillomavirus type 16-induced tumor. J. Immunother., 18: 86-94, 1995.[Medline]
  49. De Bruijn M. L., Schuurhuis D. H., Vierboom M. P., Vermeulen H., de Cock K. A., Ooms M. E., Ressing M. E., Toebes M., Franken K. L., Drijfhout J. W., Ottenhoff T. H., Offringa R., Melief C. J. Immunization with human papillomavirus type 16 (HPV 16) oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I restricted protection to HPV16-induced tumor cells. Cancer Res., 58: 724-731, 1998.[Abstract/Free Full Text]
  50. Feltkamp M. C., Vreugdenhil G. R., Vierboom M. P., Ras E., van der Burg S. H., ter Schegget J., Melief C. J., Kast W. M. Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type-16 induced tumors. Eur. J. Immunol., 25: 2638-2642, 1995.[Medline]
  51. Ressing M. E., Sette A., Brandt R. M., Ruppert J., Wentworth P. A., Hartman M., Oseroff C., Grey H. M., Melief C. J., Kast W. M. Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A0201 binding peptides. J. Immunol., 154: 5934-5943, 1995.[Abstract]
  52. Ressing M. E., van Driel W. J., Celis E., Sette A., Brandt M. P., Hartman M., Anholts J. D., Schreuder G. M., ter Harmsel W. B., Fleuren G. J., Trimbos B. J., Kast W. M., Melief C. J. Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type-16 positive cervical lesions against a human leucocyte antigen A*0201-restricted E7 encoded epitope. Cancer Res., 56: 582-588, 1996.[Abstract/Free Full Text]
  53. Nimako M., Fiander A. N., Wilkinson G. W., Borysiewicz L. K., Man S. Human papillomavirus-specific cytotoxic T-lymphocytes in patients with cervical intraepithelial neoplasia grade III. Cancer Res., 57: 4855-4861, 1997.[Abstract/Free Full Text]
  54. Santin A. D., Hermonat P. L., Ravaggi A., Chiriva-Internati M., Zhan D., Pecorelli S., Parham G. P., Cannon M. J. Induction of human papillomavirus-specific CD4+ and CD8+ lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16 and 18 positive cervical cancer. J. Virol., 73: 5402-5410, 1999.[Abstract/Free Full Text]
  55. Evans E. M., Man S., Evans A. S., Borysiewicz L. K. Infiltration of cervical cancer tissue with human papillomavirus-specific cytotoxic T-lymphocytes. Cancer Res., 57: 2943-2950, 1997.[Abstract/Free Full Text]
  56. Nakagawa M., Stites D. P., Farhat S., Sisler J. R., Moss B., Kong F., Moscicki A. B., Palefsky J. M. Cytotoxic T cell responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia. J. Infect. Dis., 175: 927-931, 1997.[Medline]
  57. Alexander M., Salgaller M. L., Celis E., Sette A., Barnes W. A., Rosenberg S. A., Steller M. A. Generation of tumor specific cytotoxic T lymphocytes from peripheral blood of cervical cancer patients by in vitro restimulation with a synthetic human papillomavirus type 16 E7 epitope. Am. J. Obstet. Gynecol., 175: 1586-1593, 1996.[CrossRef][Medline]
  58. Steller M. A., Gurski K. J., Murakami M., Daniel R. W., Shah K. V., Celis E., Sette A., Trimble E. L., Park R. C., Marincola F. M. Cell mediated immunologic responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7. Clin. Cancer Res., 4: 2103-2109, 1998.[Abstract]
  59. van Driel W. J., Ressing M. E., Kenter G. G., Brandt R. M., Krul E. J., van Rossum A. B., Schuuring E., Offringa R., Bauknecht T., Tamm-Hermelink A., van Dam P. A., Fleuren G. J., Kast W. M., Melief C. J., Trimbos J. B. Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a Phase I-II trial. Eur. J. Cancer, 35: 946-952, 1999.
  60. Ressing M. E., van Driel W. J., Brandt R. M., Kenter G. G., de Jong J. H., Bauknecht T., Fleuren G. J., Hoogerhout P., Offringa R., Sette A., Celis E., Grey H., Trimbos B. J., Kast W. M., Melief C. J. Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J. Immunother., 23: 255-266, 2000.
  61. Melnikow J., Nuovo J., Willan A. R., Chan B. K., Howell L. P. Natural history of cervical squamous intraepithelial lesions: a meta-analysis. Obstet. Gynecol., 92: 727-735, 1998.[Abstract]
  62. Puts J. J., Moesker O., de Waal R. M., Kenemans P., Vooijs G. P., Ramaekers F. C. Immunohistochemical identification of Langerhans’ cells in normal epithelium and in epithelial lesions of the uterine cervix. Int. J. Gynecol. Pathol., 5: 151-162, 1986.[Medline]
  63. Dieu M. C., Vanderbilt B., Vicari A., Bridon J. M., Oldham E., Ait-Yahia S., Briere F., Zlotnik A., LeBecque S., Caux C. Selective recruitment of mature and immature dendritic cells by distinctive chemokines expressed in different anatomic sites. J. Exp. Med., 188: 373-386, 1998.[Abstract/Free Full Text]
  64. Ressing M. E., de Jong J. H., Brandt R. M., Drijfhout J. W., Benckhuijsen W. E., Schreuder G. M., Offringa R., Kast W. M., Melief C. J. Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma. Eur. J. Immunol., 29: 1292-1303, 1999.[CrossRef][Medline]
  65. Cruz I., Meijer C. J., Walboomers J. M., Snijders P. J., Van der Waal I. Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas. Br. J. Cancer, 81: 881-889, 1999.[CrossRef][Medline]
  66. Bontkes H. J., Walboomers J. M., Meijer C. J., Helmerhorst T. J., Stern P. L. Specific MHC class I down-regulation is an early event in cervical dysplasia associated with clinical progression. Lancet, 351: 187-188, 1998.[CrossRef][Medline]



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