Identification of novel epitopes from human papillomavirus type 18 E7 that can sensitize PBMCs of multiple HLA class I against human cervical cancer

Background To identify the novel epitopes from the human papillomavirus type 18 E7 which can sensitize PBMCs of four different major HLA class I A allele. Methods Twenty-four synthetic overlapping 15-amino acid peptides were screened by measuring the frequency of CD8+ cytotoxic T lymphocytes (CTLs)-producing interferon-γ (IFN-γ) by using flow cytometry and ELISpot assays and selected peptides were validated for cytolytic activity by using the 51Cr release assay. Truncated peptides in the selected epitopes were tested to determine the important residues using ELISpot and 51Cr release assay. Results Among 24 peptides, E781-95DDLRAFQQLFLNTLS (#21) and E789-103LFLNTLSFVCPWCAS (#23) induced significantly higher Th 1 response including IFN-γ production and in vitro cytotoxicity of PBMCs of four different HLA-A alleles against cervical cancer cells than that of other peptides and the negative control (no peptide sensitization). In E781–95 (#21), amino acid position 81, 82 (N-terminus) and 92, 94, 95 (C-terminus) for HLA-A*02:02 and 24:02, and 81, 82 (N-terminus) and 92, 95 (C-terminus) for HLA-A*11:01 and 33:03 were important to elicit Th1 response of PBMCS. In E789–103 (#23), residue 100 and103 (C-terminus) were important to elicit the CD8+ CTL response in HLA-A*02:01, 11:01 and 33:03 and 100, 101, and 103 (C-terminus) were important to elicit the CD8+ CTL response in HLA-A*24:02. Conclusions E781–95 (#21) and E789–103 (#23) were identified as novel epitopes from HPV18 E7 which could sensitized PBMCs of four different HLA class I (HLA-A*02:01, 24:02, 11:01 and 33:03). These epitopes could be useful for immune monitoring and immunotherapy for HPV 18+ cervical cancer.


Introduction
A number of previous studies have reported that various types of cancer are caused by Infectious pathogens such as human papilloma virus (HPV), Helicobacter pylori, and hepatitis viruses [1,2]. HPV has been proven as a major causative pathogen of cervical cancer, anal cancer and nasopharyngeal cancer. Cervical cancer is the third most commonly diagnosed cancer and second leading cause of cancer-related death worldwide in women [3,4] and the incidence of nasopharyngeal cancer have been markedly increased during last decade in both men and women [5][6][7][8]. Therefore, the control of HPV infection is critical for the prevention or treatment of not only in precancerous states, but also in HPV-induced cancers. Among over 100 subtypes of HPV, HPV 16 and 18 are most important subtypes and about 70% of cervical cancers are associated with these two types of HPV [9].
Although currently available two prophylactic HPV vaccines have been shown to effectively prevent HPVassociated anogenital disease in young women and men [8,9], only 32% of teenagers who qualify for immunization have received all three recommended doses of the vaccine in the USA [10]. These vaccines did not show a therapeutic effect on pre-existing cervical infection or cervical lesions [11,12]. Moreover, conventional primary treatments for early stage cervical cancer showed a recurrence rate of about 15% [13,14]. In the case of late stage or recurrent disease, treatment results are relatively poor. Thus, more effective ways which can control or treat HPV infection and HPV-related cancer should be needed.
Spontaneous regression of HPV-infected precancerous lesions has been reported in patients who had strong HPV-specific Th1-biased T-cell responses [15][16][17][18]. In particularly, a strong CD8+ cytotoxic T lymphocyte (CTL) response could play an important role not only for control of HPV infection, but also for treatment of HPV-induced cervical cancer. The peptides from the HPV protein could successfully sensitize and expand HPV-specific CTLs and these peptides can be used for immune monitoring as well as for vaccine or immune therapy against HPV-induced disease [19,20]. However, one of the major drawback limiting the use of peptide-based immune monitoring or immunotherapy is that identification of at least one immune dominant epitopes from HPV molecules for each HLA class I alleles are needed for wide clinical application because CD8+ CTLs from patients or donors who have different HLA class I genetic background recognized different epitopes of HPV molecules. For overcome this drawback of peptide-based immune monitoring or immunotherapy, the identification of single peptides that can bind to multiple HLA types have been investigated and theses could lead to effective coverage of the human population by a peptide-based immune monitoring or immunotherapy. However, very few studies have addressed PBMC recognition of single peptides in a genetically heterogeneous group previously exposed to an infectious agent or cancer [21][22][23][24].
In this study, we report that novel single peptides that can bind to 4 different HLA class I which are A*02:01, A*24:02, A*11:01 and A*33:03, most frequent allele in human race. Theses single peptides were successfully elicit HPV 18-speciifc Th 1 responses of PBMCs.

Donors and peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were collected from four HLA class I (HLA-A*02:01, A*24:02, A*11:01, A*33:03) healthy donors. Human leukocyte antigen (HLA) class I genotypes were determined by the HLA laboratory at the Seoul Medical Science Institute (Seoul, South Korea) via sequence-specific polymerase chain reaction using genomic DNA. PBMCs were isolated via density gradient centrifugation using Ficoll-Hypaque 1.077 (Pharmacia Biotech, Wilkstrom, Sweden). The mononuclear cells were cryopreserved at −160°C in human AB + serum containing 10% dimethylsulfoxide (DMSO; Sigma, St Louis, MO, USA). This research was approved by the institutional review board of Yonsei University Health System, and all participants provided written informed consent.

Synthesis of HPV16 E7 peptides
A total of twenty-four, 15-amino acid peptides spanning the HPV18 E7 protein that overlapped by 11 residues, were synthesized commercially (purity of >95%; A & Pep, Yeongi-gun, South Korea; Table 1). After selection of the immune dominant candidate 15-amino acid peptides which were restricted to each HLA class I from the peptide library by screening and confirmation tests, truncated peptides spanning the candidate 15-amino acid peptides were synthesized ( Table 2). The peptides were diluted to working solution concentrations (1 μg/μL) in diethylpyrocarbonate-treated water (Invitrogen, Carlsbad, CA, USA) containing 1% DMSO and stored at −80°C before testing.

Generation of autologous dendritic cells and peptide-specific CTLs
Autologous dendritic cells (DCs) were generated as previously described, with minor modifications [25,26]. PBMCs were incubated for 2 h at 37°C using complete RPMI medium containing 10% fetal bovine serum (FBS). Adherent monocytes were resuspended at a concentration of 5 × 10 6 cells/mL in complete RPMI medium with granulocyte-macrophage colony-stimulating factor (1500 IU/mL; PeproTech, Rocky Hill, NJ, USA) and interleukin (IL)-4 (1200 IU/mL; PeproTech). On days 2, 4, and 6 of culture, fresh cytokines were added. On day 5 of culture, 10 ng/mL of tumor necrosis factor-α (R&D Systems, Minneapolis, MN, USA) was added for DC maturation. After maturation, autologous DCs were pulsed with peptides for at least 6 h. PBMCs were plated at a concentration of 2 × 10 6 cells per well in a 24-well culture plate (Nunc, Rochester, NY, USA) with 2 mL of complete RPMI medium. PBMCs were sensitized with synthetic HPV18 E7 peptides (10 μg/mL/well), and 1000 IU/mL/well of recombinant human IL-2 (rhIL-2; PeproTech) was added. Additionally, rhIL-2 (1000 IU/mL/well) was added to the culture every other day. For 1-week expansion, peptidepulsed autologous DCs (4-10 × 10 6 /well) were added to the PBMCs on day 7, incubated for 6 h, and analyzed with flow cytometry. DC-treated PBMCs were cultured for another 7 days, and cytotoxicity assays were performed.

Intracellular flow cytometric analysis
Phycoerythrin (PE)-conjugated anti-interferon-gamma (anti-IFN-γ), APC-Cy7-conjugated anti-CD44, FITCconjugated anti-CD3, PerCp-Cy5.5-conjugated anti-CD4, and APC-conjugated anti-CD8 were purchased from BD Biosciences (San Jose, CA, USA). For each sample, 1 × 10 5 events were gated on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For data analysis (Flowjo software; Tree Star, Ashland, OR, USA), positive cells were expressed as a percentage of the respective reference population. The assessment of responses was previously described in more detail [24]. Briefly, peptide-sensitized PBMCs (1 × 10 6 cells/mL) stimulated with phytohemagglutinin (PHA, Sigma) and PBMCs pulsed with autologous DCs that were not loaded with any peptide were used as positive and negative controls, respectively. One hour after stimulation, 10 μg of Brefeldin A (Sigma) was added to each well. After 5 additional hours of incubation, PBMCs were washed once with phosphate-buffered saline (PBS) and then incubated in PBS containing 1 mM ethylene-diamine-tetraacetic acid for 10 min. After 2 additional washes with PBS containing 5% FBS, the cells were incubated with fluorescently-labeled monoclonal anti-CD3, anti-CD8, anti-CD4, anti-CD44, and anti-INF-γ antibodies for 15 min on ice in the dark prior to analysis.
Plates were previously coated with anti-IFN-γ antibody (Clone AN-18, Ebioscience, San Diego, CA, USA) at 4°C, overnight. PBMCs were incubated with HPV18 E7 peptides for 48 h at 37°C and 5% CO2. Phytohemagglutinin (PHA) was added at 2.5 μg/mL as a positive control and PBMCs sensitized with no peptide were used as a negative  control. Biotinylated IFN-γ detection antibody (100 μL; 1 μg/mL in PBS) was added to each well (clone R4-6A2, Ebioscience, San Diego, CA, USA) and incubated at RT for 1.5 hours. After washing the plate 5 times with PBS, 100 μl of streptavidin-alkaline phosphatase (Invitrogen) diluted 1:1000 in PBS was added. The plate was incubated at RT for 1 h in the dark and developed with color solution by mixing 10 mL of Tris-MgCl 2 buffer with 100 μL of NBT solution (Bio-Rad, Berkeley, CA, USA) and 100 μL of BCIP solution (Bio-Rad). The plate was dried before reading. The spots were quantified with an ELISpot plate reader and software version 3.5 (AID ELISpot Reader System, Strassberg, Germany). Data were obtained by calculating the means of triplicate wells. ELISpot data were expressed as the total IFN-γ spot-forming units (SFU)/10 6 PBMCs.

Cell culture
The human HPV 18+, cervical cancer cell lines (HTB-34 for HLA-A*02:01, SNU-1160-A*2402) were purchased from by the Korean Cell Line Bank (Seoul, Korea), and it has been tested and authenticated. Cancer cell line was expanded and frozen in aliquots within 4 weeks of purchase. Cancer cells were thawed and cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA) containing 10% FBS and 1% antibiotics for no more than 8 passages. Immortalized Epstein-Barr virus-B lymphoblastoid cell lines (EBV-BLC) which were restricted each HLA class I (kindly gifted from Dr. David Stroncek, in NIH) were cultured at 37°C and 5% CO2 in RPMI-1640 with 10% FBS. Cells were routinely tested for the absence of mycoplasma.
Spontaneous and total release for each target were used to calculate the percentage of specific release according to the following formula: % specific release = (experimental counts per minutespontaneous counts per minute)/ (total counts per minutespontaneous counts per minute) × 100.

Statistical analysis
Data presented as mean ± standard error are the representative of at least 3 different experiments. To compare between control group and each tested group, a student t-test (two-tailed) was used. P-values less than 0.05 was considered statistically significant.

Discussion
A strong Th1-biased T-cell response is important for control or elimination of not only infection of HPV 16 and 18 in precancerous disease, but also HPV-induced cervical cancer. E6 and E7 protein of HPV have been suggested as good targets for immune monitoring or immunotherapy against HPV-associated disease because E6 and E7 are constitutively expressed in both HPVinfected cells and cervical cancer cells and are not express in normal cervical epithelia. Therefore, the identification of CTL specific epitopes from E6 and E7 proteins of HPV 16 and 18 is essential for the development of peptide-based immune monitoring of HPV-specific CTL response and immunotherapy in patients. However, (See figure on previous page.) Figure 5 Determination the fine specific residues within the 15-aa epitopes recognized by PBMCs using IFN-γ ELISpot assay. IFN-γ ELISpot assays were performed to determination the fine specific residues within HPV18 E7 81-95 (#21) and E7 89-103 (#23) which were recognized by PBMCs. In HLA-A*02:01 (A), residue 82 of the N-terminus and 92, 94 of C-terminus within HPV18 E7 81-95 (#21) were important to elicit Th1 response of PBMCS. Residue 100, 103 of C-terminus within E7 89-103 (#23) were important to elicit the cytotoxic response of PBMCs. In HLA-24:02 (B), residue 82 of the N-terminus and 92, 94 of C-terminus within HPV18 E781-95 (#21) were important to elicit Th1 response of PBMCS. Residue 100, 101, 103 of C-terminus within E789-103 (#23) were important to elicit the cytotoxic response of PBMCs. In HLA-A*11:01 (C) and 33:03 (D), residue 82 of the N-terminus and 94 of C-terminus within HPV18 E7 81-95 (#21) were important to elicit Th1 response of PBMCS. Residue 100, 103 of C-terminus within E7 89-103 (#23) were important to elicit the cytotoxic response of PBMCs. Data are representative of at least three independent experiments using PBMCs from HLA-A*02:01, HLA-A*24:02, HLA-A*11:01, HLA-A*33:03 subjects. Data are presented as mean ± standard error. Statistically significant differences between the tested group and negative control group were determined using a student t-test (two-tailed). **P < .001; *P < .05. a major drawback of the development of clinically effective peptide-base immune monitoring or immunotherapy is the fact that different epitopes are recognized by T cells from individuals displaying distinct major HLA molecules. Moreover, although HPV 18 is second most common subtype after HPV 16 which is associated with cervical cancer, only few CTL-specific epitopes of HPV18 have been identified. Until very recently, the studies searching for immune dominant peptides were performed testing of substantial numbers of overlapping peptides or peptide libraries. The identification of major HLA-binding motifs allowed the prediction of potential T cell epitopes (21,22), and supertypes or single peptide which can sensitize multiple HLA molecules simultaneously were found (23). However, very few studies have addressed CTL recognition of single peptide in a genetically heterogeneous group previously exposed to HPV infection or cervical cancer.
In this study, we found that two overlapping 15-amino acid peptides from HPV 18 E7 protein, E7 81-95 DDLRAFQQLFLNTLS (#21) and E7 89-103 LFLNTLSFVC PWCAS (#23), which could sensitize PBMCs of four major HLA class I A molecules including HLA-A*02:01, A*24:02, A*1101, and A*33:03, simultaneously. These four major HLA class I alleles are the most frequent HLA class I allele in human race. These 15-amino acid peptides were also successfully recognized by CD8+ CTLs of four different HLA class I alleles.
matched EBV-BLCs loaded with same truncated peptides in Figure 6.