T-cell response to human papillomavirus type 52 L1, E6, and E7 peptides in women with transient infection, cervical intraepithelial neoplasia, and invasive cancer†
An abstract of this manuscript was presented in the coming International Papillomavirus Conference at Montreal, 3–8 July 2010. Abstract No. P-118.
Abstract
The E6 and E7 proteins encoded by human papillomaviruses (HPV) are prime targets for therapeutic vaccine development. Ninety-five women with HPV 52 infection (33 transient infections, 17 cervical intraepithelial neoplasia grade II, 15 cervical intraepithelial neoplasia grade III, and 30 invasive cervical cancers) were examined for T-cell responses using interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay. Of the 29 peptides (13 L1, 10 E6, and 6 E7) screened positive by an in vitro peptide-binding assay, 14 were positive by the IFN-γ ELISPOT assay. Positive epitopes for HLA A11 were located at amino acid positions 103–111, 332–340, 342–350, and 373–381 of the L1 protein; and at 27–35 and 86–94 of the E6 protein; and at 1–9 and 27–35 of the E7 protein. A24-specific epitopes included 60–68 and 98–106 of the L1 protein, 42–50 and 59–67 of the E6 protein, and 24–32 of the E7 protein. Only one epitope (99–107) of the E6 protein showed positive responses for HLA A2 subjects. Overall, T-cell responses against L1 were observed mainly in subjects who had cleared infection; whereas responses against E6 and E7 were confined mainly to subjects who had developed cervical neoplasia. The proportion of subjects showing detectable T-cell responses was low across all grades of cervical neoplasia suggesting that immune evasion mechanisms had set on early in the course of disease progression. This study provides the first set of T-cell epitopes mapped for HPV 52, which can be considered for further evaluation as targets for immunotherapy. J. Med. Virol. 83:1023–1030, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Strong epidemiological and molecular evidence accumulated over the last decades has confirmed the association between high-risk human papillomavirus (HPV) infection and the development of cervical cancer [Franco et al., 1999; Walboomers et al., 1999; Muñoz et al., 2003]. The fact that prophylactic vaccines containing virus-like particles derived from HPV 16 and HPV 18 are highly effective in preventing cervical intraepithelial neoplasia further proves the aetiological link [Harper et al., 2006; FUTURE II Study Group, 2007; Garland et al., 2007; Paavonen et al., 2007, 2009; Muñoz et al., 2009]. Although, many countries have adopted or are considering a national immunization program, the real impact of prophylactic vaccines will only become visible at the public health level after at least a few decades [Frieden et al., 2008]. Furthermore, the cost of vaccination is still unreachable for many developing countries. It is envisaged that a substantial number of women will still suffer from cervical intraepithelial neoplasia and invasive cancer in the near future. An effective therapeutic vaccine is therefore urgently needed.
E6 and E7 are the two oncoproteins encoded by high-risk HPV. These proteins are expressed constitutively in cervical neoplasia, and therefore are potential targets for cytotoxic T-cell-mediated immune clearance. Identifying T-cell epitopes encrypted in these proteins would be crucial for the development of therapeutic vaccines. Over the last decades, studies have focused on HPV 16 which is the most frequent type found in cervical neoplasia worldwide. Recent studies have shown that there are variations in the distribution of high-risk HPV types across the world [Clifford et al., 2003; Smith et al., 2007]. HPV 52, although less common worldwide, is circulating in a higher prevalence in East Asia. HPV 52 has been detected in 19% of invasive cervical cancers in Shanghai [Huang et al., 1997], 16% in Taiwan [Ho et al., 2006], 13% in Japan [Sasagawa et al., 2001], and 9% in Hong Kong [Chan et al., 2009a]. The prevalence of HPV 52 among cervical cancers, and thus its importance, may further increase as a result of type replacement following a wide spread administration of prophylactic vaccines targeting HPV 16 and HPV 18. The objective of this study was to identify the epitopes within the HPV 52 L1, E6, and E7 proteins that can elicit T-cell responses, so as to generate data that are essential for the development of therapeutic vaccines relevant to the East Asian population where a relatively higher prevalence of HPV 52 exists.
MATERIALS AND METHODS
Study Population
Women referred to the colposcopy clinic for the management of suspected cervical lesions were recruited. All women referred consecutively, except those with immunosuppression or receiving immunosuppressive therapy, were invited to participate with informed consent. The study was approved by the local institutional ethics committee. The remainder of the cervical scrape samples collected for routine cytological examination was used for HPV detection and typing. Briefly, HPV DNA was detected by a single-round PCR based on the PGMY09/11 primers accordingly to a method described previously [Chan et al., 2009b]. Samples positive for HPV DNA were typed further by the Linear Array HPV Genotyping Kit (Roche Molecular Diagnostics, Pleasanton, CA) as described previously [Chan et al., 2009a].
The cervical disease status was confirmed by histology. Patients with no abnormalities detected by colposcopy were followed every 6 month by cytology. A transient, self-limiting HPV 52 infection was defined as having HPV 52 detected at the first visit, and became negative in subsequent visits. To fulfil the criteria for transient infection, the cytology results had to return to normal within the next 18 months.
HPV 52-positive women were invited for blood taking. The peripheral blood sample was used as a source of DNA for HLA typing. The HLA class 1A alleles of the study subjects were identified using the AccuPlex Typing kit (Dynal Biotech, Wirral, UK), which provides a low-resolution type. Briefly, genomic DNA was subjected to PCR amplification using locus-specific primers. The biotinylated PCR products were hybridized to sequence-specific oligonucleotide probes with the final signals recorded by a luminex flow analyzer. The software AccuMatch was used for HLA assignment. Subjects that belonged to the three most common HLA types (A11, A24, or A2) were invited for a further blood collection for T-cell response study using the interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay.
Screening for HPV 52 L1, E6, and E7 Peptides
Potential T-cell epitopes were predicted using the computer program RANKPEP (http://bio.dfci.harvard.edu/RANKPEP/). The results indicated that 35 peptides derived from the L1 protein (22 for HLA-A24 and 13 for HLA-A11), 26 peptides derived from the E6 protein (12 for HLA-A2, 7 for HLA-A11, and 7 for HLA-A24), and 23 peptides derived from the E7 protein (13 for HLA-A2, 5 for HLA-A11, and 5 for HLA-A24) might contain potential T-cell epitopes. These nonamers were tested by an in vitro peptide-binding assay to select possible HLA A2-, A11-, and A24-specific T-cell epitopes [Chen et al., 2009]. Briefly, the HLA-A heavy chain and the β2-microglobulin were refolded in the presence of each of the target synthetic peptides in a molecular ratio of 1:1:3. After incubation at 4°C for 24–48 hr, the soluble portion was concentrated and then analyzed by a sandwiched ELISA. Peptides showing positive results were selected for testing with the IFN-γ ELISPOT assay.
Collection of Peripheral Blood Mononuclear Cells (PBMCs)
Fifty milliliters of peripheral blood was collected from each subject. PBMCs were isolated by Ficoll-Paque™ PLUS gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Recovered PBMCs were washed, counted by Trypan blue exclusion, and cryopreserved at a cell concentration of 1 × 107 cells/ml in freezing medium containing 10% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO) and 90% fetal bovine serum (Hyclone, Logan, UT).
Interferon-γ Enzyme-Linked Immunospot (IFN-γ ELISPOT) Assay
Cryopreserved PBMCs were thawed and washed twice with AIM-V/RPMI 1640 containing 2 mM glutamine, 25 mM HEPES buffer (Invitrogen, Carlsbad, CA) supplemented with 50 µM 2-mercaptoethanol (Sigma), 0.1 µM non-essential amino acid solution (Invitrogen), and 10% human AB serum (Sigma). The concentration of viable cells was counted using Trypan blue exclusion and resuspended at 3 × 105 cells/ml. One milliliter of the PBMC suspension was added to a 24-well plate (Corning Incorporated, Corning, NY) before the addition of HLA-matched peptides at a concentration of 10 µg/ml (Table I). For the positive controls, PBMCs were cultured with HLA A2-, A11-, or A24-specific positive peptide pool containing peptides derived from Epstein–Barr virus and human cytomegalovirus at 10 µg/ml each (Table II). The PBMCs were pre-stimulated for 4 days at 37°C. Then, on day 3, 1 ml of AIM-V/RPMI 1640 medium containing 20 IU/ml recombinant IL-2 (AbD Serotec, Oxford, UK) was added. On the next day (day 4), 1 ml of the culture medium was removed and replaced with 1 ml of fresh AIM-V/RPMI 1640, and incubated for another 18–20 hr at 37°C. On day 5, peptide pulsed-PBMCs were harvested and washed. The cell concentration was adjusted to 2 × 105 cells/ml. The wells of a Multiscreen 96-well plate (Millipore Corporation, Bedford, MA) that had been pre-wetted with 35% alcohol, were washed three times with phosphate-buffered saline (Invitrogen), and coated with 100 µl of IFN-γ capture antibody (R&D Systems, Minneapolis, MN). After incubating at 4°C overnight, the plate was washed four times with phosphate-buffered saline and blocked at room temperature with 200 µl of AIM-V/RPMI 1640 medium containing 10% fetal bovine serum for at least 2 hr before PBMC seeding. PBMCs that had been pre-stimulated with the appropriate peptides were seeded in triplicates at 104 cells/well. Autologous PBMCs were added at 104 per well to serve as antigen presenting cells (APCs). Fresh peptides each at a concentration of 10 µg/ml were added to each well which contained a final volume of 100 µl of medium. For the positive controls, PBMCs that had been pulsed with HLA A2-, A11-, or A24-specific positive peptide pool were cultured again with the same set of fresh positive peptide pool containing 10 µg/ml of each peptide (Table II). PBMCs stimulated with concanavalin A (Calbiochem, Los Angeles, CA) at a concentration of 0.5 µg/ml served as a positive control for lymphocyte proliferation. Negative control wells contained only PBMCs that had gone through the 4-day incubation but without any peptide stimulation. Wells containing APCs only served as background controls. The 96-well ELISPOT plate was wrapped in aluminum foil to minimize background staining. After an incubation of 18–20 hr at 37°C, the ELISPOT plate was washed four times with 0.05% Tween-20 phosphate-buffered saline, and then 100 µl of human IFN-γ detection antibody was added (R&D Systems Inc.), and incubated at 4°C for 12–16 hr. After incubation, the washing steps were repeated and 100 µl of streptavidin-AP (R&D Systems Inc.) in 10% fetal bovine serum was added to each well. This was further incubated at room temperature for 2 hr. The plate was washed again with 0.05% Tween-20 phosphate-buffered saline, and 100 µl of BCIP/NBT chromogen (R&D Systems Inc.) was added and incubated for 30 min for color development. The reaction was stopped by washing six times with distilled water. The spots were counted using the CTL ImmunoSpot® S4 UV Analyzer (Cellular Technology Ltd., Shaker Heights, OH).
| Peptide name | HLA restriction | Protein | Amino acid positiona | Peptide sequence |
|---|---|---|---|---|
| A11-52-1 | A11 | L1 | 383–391 | STYKNENFK |
| A11-52-2 | A11 | L1 | 516–524 | SAPRTSTKK |
| A11-52-3 | A11 | L1 | 332–340 | TSESQLFNK |
| A11-52-4 | A11 | L1 | 515–523 | SSAPRTSTK |
| A11-52-5 | A11 | L1 | 373–381 | MTLCAEVKK |
| A11-52-6 | A11 | L1 | 118–126 | SFYNPETQR |
| A11-52-7 | A11 | L1 | 342–350 | LQFIFQLCK |
| A11-52-8 | A11 | L1 | 103–111 | RIKLPDPNK |
| A11-52-9 | A11 | E6 | 86–94 | KTLEERVKK |
| A11-52-10 | A11 | E6 | 36–44 | ELQRREVYK |
| A11-52-11 | A11 | E6 | 64–72 | IMCLRFLSK |
| A11-52-12 | A11 | E6 | 27–35 | RLQCVQCKK |
| A11-52-13 | A11 | E7 | 1–9 | MRGDKATIK |
| A11-52-14 | A11 | E7 | 27–35 | QLGDSSDEE |
| A11-52-15 | A11 | E7 | 90–98 | QVVCPGCAR |
| A11-52-16 | A11 | E7 | 75–83 | ATDLRTLQQ |
| A11-52-17 | A11 | E7 | 39–47 | GVDRPDGQA |
| A24-52-1 | A24 | E6 | 42–50 | VYKFLFTDL |
| A24-52-2 | A24 | E6 | 46–54 | LFTDLRIVY |
| A24-52-3 | A24 | E6 | 59–67 | PYGVCIMCL |
| A24-52-4 | A24 | E7 | 24–32 | CYEQLGDSS |
| A24-52-5 | A24 | L1 | 13–21 | YYVAGVNVF |
| A24-52-6 | A24 | L1 | 60–68 | YYYAGSSRL |
| A24-52-7 | A24 | L1 | 98–106 | QYRVFRIKL |
| A24-52-8 | A24 | L1 | 119–127 | FYNPETQRL |
| A24-52-9 | A24 | L1 | 270–278 | PYGDSLFFF |
| A2-52-1 | A2 | E6 | 45–53 | FLFTDLRIV |
| A2-52-2 | A2 | E6 | 65–73 | MCLRFLSKI |
| A2-52-3 | A2 | E6 | 99–107 | ILIRCIICQ |
- a Amino acid position numbering according to the HPV 52 prototype (GenBank accession no. X74481).
| HLA restriction | Virus_protein | Peptide sequence | Amino acid position |
|---|---|---|---|
| A2 | CMV_pp65 | VLGPISGHV | 14–22 |
| A2 | CMV_pp65 | MLNIPSINV | 120–128 |
| A2 | CMV_pp65 | NLVPMVATV | 495–503 |
| A2 | CMV_pp65 | RIFAELEGV | 522–530 |
| A11 | EBV_EBNA3B | AVFDRKSDAK | 399–408 |
| A11 | CMV_pp65 | GPISGHVLK | 16–24 |
| A11 | CMV_pp65 | ATVQGQNLK | 501–509 |
| A24 | EBV_EBNA3A | RYSIFFDY | 246–253 |
| A24 | EBV_LMP2 | TYGPVFMCL | 419–427 |
| A24 | CMV_pp65 | VYALPLKML | 113–121 |
- EBV, Epstein–Barr virus; CMV, cytomegalovirus.
The criteria for defining a positive response was adopted from published studies [van der Burg et al., 2001; Smith et al., 2005]. The net spot count for each peptide was obtained by subtracting the total number of spots observed in the test well by the average spot count of the background controls that contained only APCs. For positive T-cell responses, the mean net spot count in the peptide test wells must be greater than the mean plus 2× standard deviation (SD) of the number of spots observed from negative control wells, and the mean net spot count must be greater than 10 spots per 104 cells.
RESULTS
Study Subjects
Altogether, 95 subjects were studied for T-cell responses using the IFN-γ ELISPOT assay. These included 33 cases of transient infection with ages ranging from 33 to 68 (mean: 42.8, SD: 8.1) years, 17 cases of cervical intraepithelial neoplasia grade II with ages ranging from 33 to 68 (mean: 43.9, SD: 10.0) years, 15 cases of cervical intraepithelial neoplasia grade III with ages ranging from 33 to 55 (mean: 42, SD: 5.4) years, and 30 cases of invasive cervical cancer with ages ranging from 31 to 68 (mean: 53.7, SD: 12.3) years.
HPV 52 Infection
All 95 study subjects had HPV 52 DNA detected from their cervical scrape samples. Seventy-four subjects (23 transient infections, 13 cervical intraepithelial neoplasia grade II, 13 cervical intraepithelial neoplasia grade III, and 25 invasive cervical cancers) had HPV 52 single infection, and the other 21 (10 transient infections, 4 cervical intraepithelial neoplasia grade II, 2 cervical intraepithelial neoplasia grade III, and 5 invasive cervical cancers) had HPV 52 co-existed with one or more other HPV type. There was no significant difference in the proportion of single infection among women with different cervical status (P = 0.476 by chi-squared test).
IFN-γ ELISPOT Results
Altogether, 17 HLA A11-restricted peptides (8 L1, 4 E6, and 5 E7), nine HLA A24-restricted peptides (5 L1, 3 E6, and 1 E7), and three E6 HLA A2-restricted peptides showed positive results from the in vitro peptide-binding assay (Table I). These peptides were tested with the IFN-γ ELISPOT assay according to their HLA specificity.
Sixty-three subjects (28 transient infections, 14 cervical intraepithelial neoplasia grade II, 6 cervical intraepithelial neoplasia grade III, and 15 invasive cervical cancers) who were either homologous or heterozygous for HLA A11 were tested for the 17 HLA A11-restricted peptides. As a result, four L1 peptides located at amino acid positions 103–111, 332–340, 342–350, and 373–381 showed positive responses (Table III). The mean spot counts for L1, E6, and E7 peptides ranged from 14.0 to 34.7 per 104 cells, but no obvious patterns of distribution between spot counts among subjects with different cervical status were observed.
| HLA | Encoding region, amino acid positiona | Cervical statusb | No. with positive ELISPOT result/no. tested (%)c | Mean interferon-γ spot forming count, (range, SD)/104 cells |
|---|---|---|---|---|
| A11 | L1, 103–111 | Transient infection | 6/28 (21.4) | 29.5 (24–40, 6.8) |
| CIN2 | 0/14 (0) | — | ||
| CIN3 | 0/6 (0) | — | ||
| ICC | 0/15 (0) | — | ||
| A11 | L1, 332–340 | Transient infection | 3/28 (10.7) | 30.3 (26–35, 4.5) |
| CIN2 | 2/14 (14.3) | 17.0 (14–20, 4.2) | ||
| CIN3 | 0/6 (0) | — | ||
| ICC | 0/15 (0) | — | ||
| A11 | L1, 342–350 | Transient infection | 7/28 (25.0) | 29.9 (20–43, 7.4) |
| CIN2 | 1/14 (7.1) | 20.0 (—) | ||
| CIN3 | 0/6 (0) | — | ||
| ICC | 0/15 (0) | — | ||
| A11 | L1, 373–381 | Transient infection | 6/28 (21.4) | 34.7 (30–48, 6.8) |
| CIN2 | 2/14 (14.3) | 21.0 (18–24, 4.2) | ||
| CIN3 | 1/6 (16.7) | 22.0 (—) | ||
| ICC | 3/15 (20.0) | 18.0 (16–20, 2.0) | ||
| A11 | E6, 27–35 | Transient infection | 0/28 (0) | — |
| CIN2 | 1/14 (7.1) | 14.0 (—) | ||
| CIN3 | 1/6 (16.7) | 28.0 (—) | ||
| ICC | 0/15 (0) | — | ||
| A11 | [2,0]E6, 86–94 | Transient infection | 0/28 (0) | — |
| CIN2 | 0/14 (0) | — | ||
| CIN3 | 1/6 (16.7) | 21.0 (—) | ||
| ICC | 0/15 (0) | — | ||
| A11 | E7, 1–9 | Transient infection | 0/28 (0) | — |
| CIN2 | 1/14 (7.1) | 26.0 (—) | ||
| CIN3 | 0/6 (0) | — | ||
| ICC | 0/15 (0) | — | ||
| A11 | E7, 27–35 | Transient infection | 0/28 (0) | — |
| CIN2 | 1/14 (7.1) | 18.0 (—) | ||
| CIN3 | 0/6 (0) | — | ||
| ICC | 0/15 (0) | — | ||
| A11 | Positive peptide pool | 82.1 (45–145, 33.1) | ||
| A24 | L1, 60–68 | Transient infection | 2/7 (28.6) | 17.5 (15–20, 3.5) |
| CIN2 | 1/4 (0) | — | ||
| CIN3 | 0/7 (0) | — | ||
| ICC | 0/14 (0) | — | ||
| A24 | L1, 98–106 | Transient infection | 3/7 (42.9) | 36.7 (33–42, 4.7) |
| CIN2 | 1/4 (25.0) | 38.0 (—) | ||
| CIN3 | 0/7 (0) | — | ||
| ICC | 0/14 (0) | — | ||
| A24 | E6, 42–50 | Transient infection | 0/7 (0) | — |
| CIN2 | 1/4 (25.0) | 18.0 (—) | ||
| CIN3 | 0/7 (0) | — | ||
| ICC | 0/14 (0) | — | ||
| A24 | E6, 59–67 | Transient infection | 1/7 (14.3) | 18.0 (—) |
| CIN2 | 1/4 (25.0) | 25.0 (—) | ||
| CIN3 | 0/7 (0) | — | ||
| ICC | 0/14 (0) | — | ||
| A24 | E7, 24–32 | Transient infection | 0/7 (0) | — |
| CIN2 | 1/4 (25.0) | 14.0 (—) | ||
| CIN3 | 0/7 (0) | — | ||
| ICC | 2/14 (14.3) | 19.5 (16–23, 4.9) | ||
| A24 | Positive peptide pool | 49.5 (30–98, 22.4) | ||
| A2 | E6, 99–107 | Transient infection | 0/20 (0) | — |
| CIN2 | 0/10 (0) | — | ||
| CIN3 | 1/8 (12.5) | 26.0 (—) | ||
| ICC | 0/16 (0) | — | ||
| A2 | Positive peptide pool | 107.6 (76–159, 29.8) |
- a Amino acid position numbering according to the HPV 52 prototype (GenBank accession no. X74481).
- b CIN, cervical intraepithelial neoplasia; ICC, invasive cervical cancer.
- c Subjects with heterozygous HLA A alleles were tested for each of the HLA type, and therefore counted twice.
Positive responses to L1 peptides were mainly observed from subjects with transient infection, and with positive response rates ranged from 10.7% to 25.0%. Three of the four L1 peptides also elicited positive responses from subjects with cervical intraepithelial neoplasia grade II (positive rates ranged from 7.1% to 14.3%). In contrast, only one L1 peptide (373–381) could elicit positive responses from subjects with cervical intraepithelial neoplasia grade III and invasive cervical cancer (Table III).
A different pattern of response was observed for E6 peptides. The E6 peptide located at amino acid position 27–35 showed positive responses for subjects with cervical intraepithelial neoplasia grade II (7.1%) and cervical intraepithelial neoplasia grade III (16.7%), and the other E6 peptide (86–94) showed positive responses for one (16.7%) subject with cervical intraepithelial neoplasia grade III. None of the E6 peptides had elicited positive responses from subjects with transient infection. The results of E7 peptides were similar to those of E6. Two E7 peptides (1–9 and 27–35), each showed positive results from one subject with cervical intraepithelial neoplasia grade II (Table III).
Nine HLA A24-restricted peptides (5 L1, 3 E6, and 1 E7) were tested against 32 subjects (7 transient infections, 4 cervical intraepithelial neoplasia grade II, 7 cervical intraepithelial neoplasia grade III, and 14 invasive cervical cancers) who were either homozygous or heterozygous for the HLA A24 allele. Two L1 peptides located at amino acid positions 60–68 and 98–106 showed positive responses with mean spot counts ranged from 17.5 to 38.0 per 104 cells. The L1 60–68 peptide elicited positive responses from two (28.6%) women with transient infection; whereas the L1 98–106 showed positive results from three (42.9%) women with transient infection, and from one (25%) subject with cervical intraepithelial neoplasia grade II. None of these L1 peptides had elicited positive responses from subjects with cervical intraepithelial neoplasia grade III or invasive cervical cancer (Table III). Of the two positive E6 peptides, one (E6 42–50) showed positive responses from a case of cervical intraepithelial neoplasia grade II, and the other peptide (E6 59–67) showed positive responses from a case of transient infection and a case of cervical intraepithelial neoplasia grade II, respectively (Table III). Only one E7 peptide (24–32) showed positive responses, and was observed from a case of cervical intraepithelial neoplasia grade II and two cases of invasive cervical cancer.
Three A2-restricted peptides were tested with PBMCs collected from 54 subjects (20 transient infections, 10 cervical intraepithelial neoplasia grade II, 8 cervical intraepithelial neoplasia grade III, and 16 invasive cervical cancers). One of the peptides, E6 99–107, showed positive responses (26 spots per 104 cells) for a woman with cervical intraepithelial neoplasia grade III.
DISCUSSION
The evidence for an etiological association between HPV and the development of cervical cancer is beyond doubt [Muñoz, 2000; zur Hausen, 2002]. The potential benefits of implementing HPV vaccination could well be more than decreasing the health burden associated with cervical or genital diseases, as evidence that HPV infection also plays a role in the development of cancers in other anatomical locations, especially the head and neck region, is accumulating [Gillison et al., 2008; Giuliano et al., 2008; zur Hausen, 2009]. While two highly effective prophylactic vaccines are available currently [Harper et al., 2006; FUTURE II Study Group, 2007; Garland et al., 2007; Paavonen et al., 2007, 2009; Muñoz et al., 2009], the development of a therapeutic vaccine remains at the stage of early clinical trials, and with no licensed vaccines available for clinical use [Muderspach et al., 2000; Smith et al., 2005; Hung et al., 2007; Alvarez-Salas, 2008; Michelin and Murta, 2008; Welters et al., 2008; Trimble et al., 2009].
HPV shows a distinct pattern of protein expression according to the state of differentiation of the host cell. The late structural proteins L1 and L2 are expressed during the lytic vegetative cycle, and are found mainly in the upper most layers of a differentiated epithelium. The early, transforming proteins E6 and E7 are expressed mainly in the lower layer of undifferentiated cells. In a transformed tumor cell, the E6 and E7 proteins are expressed constitutively, and are thought to be essential for maintaining the malignant phenotype [zur Hausen, 2002; Doorbar, 2006]. E6 and E7 are therefore prime targets for the development of therapeutic vaccines. The clearance of an established virus-infected lesion is likely to require both CD4+ and CD8+ T-cell responses against HPV. Identifying T-cell epitopes from the E6 and E7 proteins will therefore be crucial for developing a successful therapeutic vaccine.
Since HPV 16 is the most prevalent type found in cervical cancers worldwide, most of the current data on CD8+ T-cell epitopes have been derived from HPV 16 and based on HLA A2-positive subjects [Evans et al., 1996; Ressing et al., 1996; Nimako et al., 1997; Bontkes et al., 2000; Valdespino et al., 2005]. This study provided data on another HPV type, HPV 52, which exists in a relatively high prevalence in East Asia. Furthermore, this study focused on the HLA alleles that are more prevalent in the Asian population.
The proportion of subjects showing HPV-specific T-cell responses were generally low across different degrees of cervical neoplasia. The low-positive rate observed might due to the use of single epitope rather than mixtures containing every conceivable epitope. Nevertheless, the observed results are in line with previous reports of low CD8+ T-cell responses even when using full-length proteins or long peptides encompassing the entire E6 or E7 of HPV 16 [Evans et al., 1996; Ressing et al., 1996; Nimako et al., 1997; Bontkes et al., 2000; Woo et al., 2010]. Of note, a recent report has demonstrated memory cytotoxic T-cell responses to HPV E6/E7 peptides in all cervical cancer patients examined [Valdespino et al., 2005].
Although the number of subjects recruited in the current study was relatively small, the data suggested that the proportion of subjects showing positive responses were higher for cervical intraepithelial neoplasia grade II compared to cervical intraepithelial neoplasia grade III or invasive cervical cancer. This finding suggests that immune evasion mechanisms may exert a greater effect as the virus-infected lesion progresses. This might result from changes caused by integration of the HPV genome [Pett et al., 2004]. It has been shown that the decrease in anti-tumor immunity in cancer patients was in general associated with the increase in regulatory T cells. This phenomenon has also been observed in patients with cervical cancer, and the presence of CD4+ regulatory T cells was associated with persistent infection and disease progression [Molling et al., 2007; Visser et al., 2007; van der Burg et al., 2007; Narayan et al., 2009].
It is likely that at the stages of cervical intraepithelial neoplasia grade III and invasive cancer, the number of circulating memory CD8+ T cells have decreased to very low levels that can only be detected by extremely sensitive methods. Nevertheless, there is still a potential to stimulate the proliferation of these clones of E6/E7-specific memory T cells by appropriate antigens, and to activate T-cell effector function against the transformed tissue. Interestingly, stronger T-cell responses against E6 and E7 have been observed upon the depletion of regulatory T cells [Visser et al., 2007]. The therapeutic potential of peptide-based vaccines is further supported by a recent clinical trial on vulvar neoplasia [Kenter et al., 2009].
In addition to E6 and E7 peptides, this study also examined T-cell responses to L1 peptides. Majority of the positive responses toward L1 peptides were observed from subjects who had recently cleared an HPV 52 infection. In contrast, only a few subjects with cervical intraepithelial neoplasia grade II mounted responses against the L1 peptides. This may suggest that the L1 cellular responses were short-lived and disappeared when L1 protein is no longer expressed in the lesion. The value of using L1 as a therapeutic vaccine target is uncertain at this stage.
In summary, the current study has identified several epitopes located at the L1, E6, and E7 proteins of HPV 52. These include eight HLA A11-specific epitopes (L1: 103–111, 332–340, 342–350, and 373–381; E6: 27–35 and 86–94; and E7: 1–9 and 27–35), five A24-specific epitopes (L1: 60–68 and 98–106, E6: 42–50 and 59–67, and E7: 24–32), and one HLA A2-specific E6 epitope located at amino acid position 99–107. It would be worthwhile to evaluate whether these epitopes could be targets for the development of therapeutic vaccines tailored especially for the East Asian population where HPV 52 infection is prevalent [Chan et al., 2006; Bao et al., 2008; Bhatla et al., 2008]. There is an urgent need to develop vaccines targeting HPV 52 as its share of disease burden will increase with the control of HPV 16 and HPV 18 by current vaccines.
