Skip to main content
NCBI home page
Clinical and Vaccine Immunology : CVI logoLink to Clinical and Vaccine Immunology : CVI
. 2013 Jul;20(7):1027–1033. doi: 10.1128/CVI.00721-12

Cytolytic Activity of the Human Papillomavirus Type 16 E711-20 Epitope-Specific Cytotoxic T Lymphocyte Is Enhanced by Heat Shock Protein 110 in HLA-A*0201 Transgenic Mice

Zhenzhen Ding a,b, Rongying Ou c, Bing Ni d, Jun Tang e,✉, Yunsheng Xu a,✉
PMCID: PMC3697441  PMID: 23658393

Abstract

Heat shock proteins (HSPs) have been successfully applied to a broad range of vaccines as biological adjuvants to enhance the immune response. The recently defined HSP110, in particular, exhibits strong protein binding affinity and is capable of enhancing the immunogenicity of protein antigens remarkably more than other HSP family members. In our previous study, we verified that murine HSP110 (mHSP110) significantly enhanced the immune response of a C57BL/6 mouse model to the H-2d-restricted human papillomavirus (HPV) E749-57 epitope (short peptide spanning the 49th to 57th amino acid residues in the E7 protein). To determine whether HSP110 similarly enhances the immunogenicity of human epitope peptides, we used the HLA-A2 transgenic mouse model to investigate the efficacy of the mHSP110 chaperone molecule as an immunoadjuvant of the human HLA-A2-restricted HPV16 E711-20 epitope vaccine. Results showed that mHSP110 efficiently formed a noncovalently bound complex with the E711-20 epitope. The mHSP110-E711-20 complex induced epitope-specific splenocyte proliferation and E711-20-specific gamma interferon (IFN-γ) secretion. Importantly, cytotoxic T lymphocytes primed by the mHSP110-E711-20 complex exerted strong cytolytic effects on target T2 cells pulsed with the E711-20 peptide or TC-1 cells transfected with the HLA-A2 gene. In addition, the mHSP110-E711-20 complex elicited stronger ex vivo and in vivo antitumor responses than either emulsified complete Freund's adjuvant or HSP70-chaperoned E711-20 peptide. These collective data suggest that HSP110 is a promising immunomodulator candidate for peptide-based human cancer vaccines, such as for the HLA-A2-restricted E711-20 epitope.

INTRODUCTION

Cervical cancer (CaCx) is the second leading cause of cancer deaths among women worldwide. CaCx is strongly associated with human papillomavirus (HPV) infection, particularly HPV types 16 and 18. Several clinical studies have demonstrated constitutive expression of the HPV16 oncoproteins E6 and E7 in the majority of cervical tumor cells (1–4). Since cervical tumors often recur after surgery and/or radiotherapy (5, 6), it is critical to develop prophylactic and therapeutic strategies that completely eliminate the tumor cells. HPV16 cytotoxic T lymphocyte (CTL) epitopes may be good candidates for the development of an effective peptide-based antitumor vaccine (7–9). In fact, studies in animal models have already shown that HPV16-specific CTLs can safely eradicate HPV16-transformed tumor cells in vivo (10).

To date, many therapeutic vaccine strategies have been developed targeting the HPV16 E6/E7 proteins (11–14). Among these, the synthetic peptide-based strategies have the particularly remarkable advantages of convenient and low-cost high-throughput preparation and rare occurrences of adverse reactions, including general toxicity, immunosuppression, and autoimmunity (15–17). More importantly, when generating specific CTLs it is possible to select the length of the amino acid sequence that is most suitable for the particular application. However, peptide-based vaccines are characterized by weak immunogenicity when used alone in vivo (18). To overcome this limitation, it is critical to identify adjuvant vehicles that can enhance the immunogenicity of peptides but are not specifically immunogenic themselves. A large number of immune adjuvants have been investigated for this purpose, including aluminum hydroxide, liposomes, cytokines (such as granulocyte-macrophage colony-stimulating factor [GM-CSF] and interleukin 2 [IL-2]), saponins, Toll-like receptor (TLR) agonists, and various heat shock proteins (HSPs) (19–21). Among these, the HSPs have shown especially promising results and are being heavily pursued as tools with which to develop effective and safer CaCx vaccines.

It has long been established that HSPs, particularly HSP70, can function as effective immunoadjuvants by virtue of their ability to bind tumor-specific peptides. However, recent research has indicated that a larger HSP, HSP110, is better suited for use in recombinant vaccines (22, 23). HSP110 is a major HSP of eukaryotic and mammalian cells in general. With a molecular mass of 110 kDa, HSP110 is composed of four functionally coupled domains, including the N-terminal domain that mediates ATP binding and the peptide-binding domain (amino acid residues 394 to 509) (24). Several studies have suggested that HSP110 can efficiently bind large proteins and peptides under heat shock conditions and that it produces excellent adjuvant effects (25–28).

The mouse H2-Db-restricted CTL epitope, E749-57 (short peptide spanning the 49th to 57th amino acid residues in the E7 protein; RAHYNIVTF), was previously shown to form a noncovalent binding complex with HSP110 at a 1/50 molar ratio and to elicit antitumor immunity in C57BL/6 mice (29). Moreover, the epitope E711-20 is not only a well-known HLA-A*0201-restricted human CTL epitope of the HPV16 E7 protein that shows high-affinity binding to HLA-A2 in vitro (8, 30, 31). In addition, its ability to induce peptide-specific CTLs has been verified in HLA-A2 transgenic mice (32). HLA-A2 is the most frequent HLA-A specificity in the human population, with an allele frequency of 10 to 40% in different ethnic groups (33). Specifically, about 50.7% of random individuals in the population in China carry HLA-A2, and HLA-A*0201 ranks first in frequency (allele frequency, 15.5%) (34). Thus, peptide vaccines based on this haplotype may have a particularly potent impact on the global HPV threat.

In the study presented herein, we investigated whether the CTL response induced by epitope E711-20 can be enhanced by an HSP110 chaperone molecule. We found that murine HSP110 (mHSP110) can combine with E711-20 under heat shock conditions. Application of this novel strategy in an HLA-A*0201 transgenic mouse model validated that the mHSP110-E711-20 complex induces functional cytotoxic T cells in a peptide-specific manner and does so more efficiently than the complete Freund's adjuvant (CFA)-emulsified peptide E711-20 or HSP70 alone.

MATERIALS AND METHODS

Mice, plasmids, and cell lines.

HLA-A*0201 transgenic mice (females, 4 to 8 weeks old) expressing HLA-A2.1 on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) (35). All mice were housed in a pathogen-free environment and all procedures involving the animals were carried out with approval from the local ethics committee of the Third Military Medical University. TC-1 tumor cells, derived from primary pulmonary epithelial cells of C57BL/6 mice cotransformed with HPV16 E6 and E7 and c-Ha-ras oncogenes (36), were purchased from the Cell Center at the CAMS in Shanghai, China. The eukaryotic expression plasmid HLA-A2/Kb expressing the α1 and α2 domains of HLA-A*0201 and the α3 domain of H-2Kb (plasmid number 14906; Addgene, Cambridge, MA) was transfected into the TC-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and maintained in complete RPMI 1640 medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 2 mM l-glutamine (Gibco), 50 mM 2-mercaptoethanol (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO2 in air (37). To establish stable A2Kb-expressing TC-1/A2Kb cells, the cells were selected by addition of 500 μg/ml G418 (Sangon, Shanghai, China) to the complete RPMI 1640 medium for about 3 weeks until the control cells were dead. T2 cells, an HLA-A2-positive and antigen-processing-defective cell line, were obtained from the American Type Culture Collection (Manassas, VA) and maintained as previously described (35).

Synthetic peptides.

The HPV E711-20 peptide epitope (YMLDLQPETT) was synthesized with a free carboxy terminus by using solid-phase strategies on an automated multiple peptide synthesizer (Shenzhen Hanyu Pharmaceutical Corp., Shenzhen, China) with 9-fluorenylmethoxy carbonyl (Fmoc) chemistry. The purity of all synthesized peptides was >90% as detected by reverse-phase high-performance liquid chromatography (HPLC). Synthesized peptides were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Fluorescein isothiocyanate (FITC)-labeled E711-20 peptides (FITC conjugation at the N terminus; 90% pure) were synthesized (Shenzhen Hanyu Pharmaceutical Corp.) and dissolved in phosphate-buffered saline (PBS) with 1% DMSO and stored at −20°C.

Expression, purification, and identification of recombinant proteins.

mHSP110 cDNA was cloned into the pQE-80L vector (Qiagen, Hilden, Germany) and transformed into recombinant Escherichia coli M15 and cultured in Luria broth. After induction by addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), the mHSP protein with 6×His tags was purified from the culture supernatant using an Ni-nitrilotriacetic acid (Ni-NTA) column, which was washed extensively with 1% Triton X-100/6 M guanidine-HCl to remove endotoxins, as described previously (29). The lipopolysaccharide (LPS) in the final protein solution was undetectable by a Limulus test. The prepared mHSP110 and commercial source mHSP70 (LPS free; Cusabio, Wuhan, China) were stored at −70°C. Concentrations of the recombinant proteins were measured by Bradford assay. Protein purity was assessed with an SDS-PAGE assay with Coomassie blue staining. Western blot analysis was used to confirm the identity of the purified recombinant protein with rabbit anti-HSP110 or anti-mHSP70 polyclonal antibody (Stressgen, Ann Arbor, MI) as the primary antibody, and a chemiluminescence kit (Roche Diagnostics Limited, Shanghai, China) was used to detect the target protein band, followed by exposure to X-ray film.

Reconstitution and confirmation of HSP110-HPV E711-20 peptide complex.

The noncovalent complex of mHSP110-E711-20 was generated according to the previously published protocol for binding mHSP110 with peptide E749-57 (29). Briefly, mHSP110 and E711-20 or FITC-labeled E711-20 peptide were mixed at a 1:50 molar ratio in PBS supplemented with 1 mM ATP and 1 mM MgCl2. After 30 min of incubation at 50°C, the samples were cooled to room temperature for 1 h. Free peptides were cleared from the sample by a size exclusion filtration method using a Microcon 50 filter (Millipore, Billerica, MA) with a molecular mass cutoff of 50 kDa. The remaining bound complexes of E711-20/mHSP110-FITC were evaluated by native PAGE (NuPAGE Novex Tris-acetate minigels; Invitrogen), which was carried out in a darkroom. The gel was observed and photographed on a fluorescence imager and then stained with Coomassie brilliant blue R250. The PageRuler prestained protein ladder (Fermentas, Vilnius, Lithuania) was used to determine protein and complex sizes.

Immunization, cell purification, and culturing.

Mice were divided into groups of five and administered one of the following immunogens via the intraperitoneal (i.p.) route: mHSP110-E711-20 complex (100 μg mHSP110 plus 50 μg E711-20), mHSP70-E711-20 complex (100 μg mHSP70 plus 50 μg E711-20), 100 μg of mHSP110 alone, 100 μg of mHSP70 alone, 50 μg of E711-20 alone, or 100 μl PBS. All mice were immunized twice, with the initial immunization occurring on day 0 and the booster given 14 days later. Another group of five mice were immunized subcutaneously (s.c.) with 50 μg of E711-20 together with CFA and then boosted with 50 μg of E711-20 together with incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO).

All immunizing agents were dissolved in PBS to achieve a final 100-μl volume. For all immunization groups, the mice were euthanized 2 weeks after the second vaccination. The spleens were aseptically removed and made into single-cell splenocyte suspensions. Red blood cells were removed by treating the suspension with red blood cell lysis buffer (Roche Diagnostics Limited). The remaining splenocytes were then washed in PBS containing 5% FBS and cultured at 3 × 106/ml in 24-well culture plates with complete T-cell medium (RPMI 1640 containing 10% FBS, 2 mM l-glutamine, penicillin [100 U/ml], streptomycin [100 μg/ml], and 5 × 10−5 M 2-mercaptoethanol) at 37°C in 5% CO2. The cultured cells were then subjected to various assays, including lymphocyte proliferation, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), and cytotoxicity.

Splenocyte proliferation assay.

Splenocytes from the various groups of immunized mice were cultured (2 × 104 cells/well, 100 μl) in triplicate in 96-well culture plates in the presence of the E711-20 peptide for 5 days at 37°C in 5% CO2. The number of viable cells was then determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test kit (Sigma-Aldrich).

Cytotoxicity assay.

Splenocytes (1 × 107 in RPMI 1640 containing 10% FBS) from the various groups of immunized mice were stimulated in vitro with E711-20 (10 μg/ml) and murine interleukin 2 (mIL-2) (10 U/ml) (R&D Systems, Minneapolis, MN). At day 5 of culture, the viable splenocytes were used as effector cells. Meanwhile, T2 cells were preincubated with peptide E711-20 (10 μg/ml) for 4 h, washed in RPMI 1640 containing 5% FBS, and then used as the target cells of the stimulated splenocytes. T2 cells alone were used as the target cell control. Furthermore, to test whether the T cells from mice that were immunized with immunogens recognize the E711-20 epitope naturally processed from E7 protein, the TC-1/A2Kb and TC-1 cells were applied as the target cells.

The CTL activity of the effector cells was measured using a nonradioactive cytotoxicity kit (Promega, Madison, WI) as previously described (29). Briefly, the stimulated splenocytes were cocultured with 5 × 104 target cells at different effector/target cell (E/T) ratios of 50:1, 25:1, and 12.5:1 in 96-well V-bottom culture plates at 37°C in 5% CO2. After 4 h of incubation, the supernatants (50 μl/well) were removed and the substrate mix (50 μl/well) was added. The plates were incubated in the dark for 30 min at room temperature, after which the reaction was terminated by adding 50 μl of stop solution (1 M acetic acid). Sample absorbance at a 490-nm wavelength was measured by an ELISA plate reader (Bio-Rad, Hercules, CA).

Spontaneous release of lactate dehydrogenase (LDH) by the target or effector cells was assessed by incubating the target cells in the absence of effector cells and vice versa. The maximum release of LDH was determined by incubating target cells in 1% Triton X-100 in assay medium. The specific lysis percentage was calculated as [(experimental release − effector spontaneous release − target spontaneous release)/(target maximum release − target spontaneous release)] × 100. All experiments were repeated three times.

ELISA.

Levels of gamma interferon (IFN-γ) in cell culture supernatants were determined by using a mouse IFN-γ-precoated ELISA kit (R&D Systems) according to the manufacturer's instructions. Briefly, the splenocyte supernatants (100 μl/well) were harvested on day 7 of culture and were added to the precoated wells. After incubation at room temperature for 2 h and washing 4 to 6 times, biotinylated antibody (100 μl/well) was added and the plates were incubated for 1 h at 37°C. After an additional 15-min incubation with enzyme conjugate at 37°C, the reaction was stopped by adding 100 μl of stop solution and the optical density was read at 450 nm. Three independent experiments were carried out for each sample.

ELISPOT assay.

Antigen-specific IFN-γ-secreting T cells were detected by a mouse IFN-γ-precoated ELISPOT kit (Dakewei, Shenzhen, China) according to the manufacturer's protocol. Briefly, the precoated plate was activated by adding Lympho-Spot serum-free medium. Splenocytes (5 × 105 cells/well, 100 μl) were added in triplicate wells and incubated in the presence of either E711-20 (10 μg/ml) or HSP110 (10 μg/ml) at 37°C in 5% CO2 for 36 h. Positive control wells (1 × 105 cells/well) were established with phytohemagglutinin alone (20 μg/ml). Wells containing only Lympho-Spot serum-free medium were established to measure the background signal. Then, ddH2O (100 μl/ml) was added to the wells and the plates were incubated for 10 min at 4°C. After extensive washing (5 to 7 times), biotinylated IFN-γ antibody (Ab) (100 μl/well) was added and the plates were incubated for 1 h at 37°C. After five more washes, streptavidin horseradish peroxidase (HRP) (100 μl/well) was added and the plates were incubated for 1 h at room temperature. After a final five washes, the immunoreactive spots were developed by incubating with aminoethyl carbazole (AEC) chromogenic solution (100 μl/well) for 25 min at room temperature. The spots were counted using a dissecting microscope.

Tumor challenge experiments.

To evaluate the therapeutic antitumor effects of the various immunogens, tumors were established by s.c. injection of 5 × 105 TC-1/A2Kb cells into the right flank of HLA-A*0201 transgenic mice (day 0) (37, 38). On day 5, mice with obvious tumor development were assigned arbitrarily into 8 groups of 10. Mice were then i.p. injected with different immunization formulations (150 μl total volume in PBS) on days 5, 9, and 14. Tumor volume was recorded twice a week by measuring perpendicular tumor diameters with a caliper and calculating (the shortest diameter2 × the longest diameter)/2. Survival of mice was recorded daily for each group.

Statistical analysis.

All data are expressed as mean ± standard error of the mean (SEM). Comparisons between experimental groups were made using Student's t test and analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Differences were considered statistically significant if the P value of the difference was <0.05.

RESULTS

Noncovalent binding of mHSPs to E711-20 under heat shock conditions.

Recombinant mHSP110 was prepared as described in our previous study (29). The recombinant mHSP110 protein preparation yielded high purity, as shown in the SDS-PAGE assay (Fig. 1, lanes 1 to 3). The purified mHSP110 and the commercial source mHSP70 were further confirmed by Western blotting (Fig. 1, lanes 4 and 6). Prior studies in our laboratory indicated that mHSP110 efficiently binds the HPV16 epitope peptide E749-57 (RAHYNIVTF) at heat shock temperature (29). Herein, to determine whether the mHSP110 and mHSP70 can also bind the E711-20 peptide, the FITC-labeled E711-20 peptides were incubated with mHSP110 or mHSP70 in the binding buffer under heat shock conditions, and the complex was then subjected to 10% nondenaturing PAGE analysis and imaged under a fluorescence imager. As shown in lanes 5 and 7 of Fig. 1, an FITC signal was detected at the position corresponding to the mHSP110 or mHSP70 protein in the gel, indicating that mHSP110 and mHSP70 could complex with the FITC-E711-20 peptides. However, when the complex was run on an SDS-PAGE gel, no fluorescent signal was observed at the position corresponding to the mHSP110 band (data not shown), which indicated noncovalent binding of mHSP110 with the peptide E711-20.

Fig 1.

Fig 1

Open in a new tab

Verification of the mHSP110-E749-57 and mHSP70-E749-57 complex. SDS-PAGE resolution of the cell culture supernatants before (lane 1) and after (lane 2) IPTG induction and of the purified mHSP110 (lane 3). Western blot detection of the purified mHSP110 (lane 4) and the commercial source mHSP70 (lane 6). Nondenaturing gradient PAGE assay detection of the HSP70 protein (lane 5) and the purified mHSP110 protein mixed with FITC-labeled E749-57 (lane 7) under heat shock conditions by imaging with a fluorescence imager (lanes 5 and 7). M, protein molecular mass markers.

mHSPs-E711-20 complexes promoted antigen-specific splenocyte proliferation.

After HLA-A*0201 transgenic mice were immunized with the various immunogens, their splenocytes were harvested and restimulated in vitro with the E711-20 peptides for 5 days. MTT assay showed that the mice immunized with the mHSP110-E711-20 complex exhibited the strongest lymphocyte proliferation compared with all other groups (Fig. 2). The mHSP70-E711-20 complex and the CFA-emulsified E711-20 peptide primed similar lymphocyte proliferation levels, both of which were markedly higher than those observed in the E711-20, mHSP110, and mHSP70 groups. In contrast, the spleen cells from mice immunized with mHSP110, mHSP70, and E711-20 did not stimulate significant cell proliferation after in vitro restimulation, as evidenced by the indexes not being significantly different from that detected in the PBS negative-control group.

Fig 2.

Fig 2

Open in a new tab

Proliferation of splenocytes from mice immunized with the indicated immunogens. Data represent mean ± SEM of three independent experiments. #, P < 0.01 compared to all other groups; *, P < 0.05 compared to the E711-20, mHSP110, mHSP70, or PBS groups.

mHSP110-E711-20 chaperone complex stimulates enhanced IFN-γ secretion in immunized mice.

To determine whether the mHSP110-E711-20 complex stimulated the cellular immune response, IFN-γ production by splenocytes of the immunized mice was measured by using the ELISA method. HLA-A*0201 transgenic mice were immunized twice with the indicated immunogens, with a 2-week interval between immunizations. Two weeks after the second immunization, splenocytes harvested from the immunized mice were stimulated with E711-20 in vitro, as described above. As shown in Fig. 3A, significantly increased IFN-γ production was observed in the supernatants of cultured splenocytes from mice vaccinated with the mHSP110-E711-20 chaperone complex compared to the supernatants of cells from mice vaccinated with all other immunogens. However, the mHSP70-E711-20 complex and the CFA-emulsified E711-20 peptide stimulated much higher IFN-γ production than the immunogens E711-20, mHSP110, and mHSP70 or the PBS control.

Fig 3.

Fig 3

Open in a new tab

Enhanced IFN-γ production elicited by the indicated immunogens in HLA-A*0201 transgenic mice. (A) IFN-γ in the splenocyte culture supernatants measured by ELISA. (B) IFN-γ-producing splenocytes detected by ELISPOT assay. #, P < 0.01 compared to all other groups; *, P < 0.05 compared to the mHSP110, mHSP70, E711-20, or PBS groups.

Compared with the ELISA method, the ELISPOT assay is a sensitive functional method for measuring IFN-γ production at the single-cell level. Therefore, ELISPOT was employed to further investigate whether the HSP110-E711-20 complex elicited antigen-specific IFN-γ production. In accordance with the ELISA results, the HSP110-E711-20-immunized mice primed the most IFN-γ-producing cells, compared with all other groups (Fig. 3B). Again, the mHSP70-E711-20 complex and the CFA-emulsified E711-20 peptide primed more IFN-γ-producing cells than mHSP110, mHSP70, and E711-20 alone or the PBS control (Fig. 3B).

mHSP110-E711-20 complex primed potent and specific cytotoxicity.

We next used an LDH release assay to investigate whether the mHSP110-E711-20 complex elicited the antigen-specific CTLs from HLA-A*0201 transgenic mice immunized with the mHSP110-E711-20 complex and several control agents. T2 cells pulsed with the E711-20 peptide were used as the target cells, because T2 cells express HLA-A2 molecules but do not process intracellular antigens. As shown in Fig. 4A, in the mice vaccinated with the mHSP110-E711-20 complex, the cytolytic activity of effector T cells on the T2 cells pulsed with the E711-20 peptide was significantly higher than in other groups when the E/T ratio was 50:1. At this ratio, however, both mHSP70-E711-20 and CFA-E711-20 stimulated higher cytotoxicity than E711-20 alone, mHSP110 alone, mHSP70 alone, and the PBS control. When the untreated T2 cells were used as target cells to investigate whether natural killer (NK) cells contribute to the nonspecific cytotoxicity in this assay, no apparent difference of cytotoxicity was observed between any of the groups (Fig. 4B).

Fig 4.

Fig 4

Open in a new tab

Epitope-specific CTL response elicited by the indicated immunogens in HLA-A*0201 transgenic mice. CTL cytolytic activity was assessed by LDH assay of T2 cells pulsed with the E711-20 peptide (A), T2 cells alone (B), HLA-A2Kb-expressing plasmid-transfected TC-1 cells (C), or TC-1 cells alone (D) as the target cells. Data represent mean ± SEM of three independent experiments.

We further investigated whether the CTLs from immunized HLA-A*0201 transgenic mice recognized the E711-20 epitope naturally processed from the E7 protein by using TC-1/A2Kb as the target cells and TC-1 cells as the control target cells. The results were similar to those obtained when the E711-20-loaded T2 cells were used as the target cells (Fig. 4C). Likewise, none of the cytotoxic T cells from any of the groups killed the TC-1 cells (Fig. 4D).

Potent in vivo antitumor effect of immunization with mHSP110-E711-20 complex.

Therapeutic efficacy against established tumors in vivo is the most important indication for the potential application of vaccines. Thus, to determine whether the enhanced ex vivo E7-specific T cell response generated by the mHSP110-E711-20 complex elicited therapeutic antitumor effects, we performed in vivo tumor treatment experiments using the HLA-A2Kb/TC-1 tumor model by s.c. inoculation of HLA-A2Kb-expressing TC-1 cells into HLA-A*0201 transgenic mice. As shown in Fig. 5A, although the mHSP70-E711-20 and CFA-emulsified E711-20 immunizations inhibited tumor growth, the tumor-bearing mice treated with the mHSP110-E711-20 complex exhibited a stronger decrease in tumor growth than the tumor-bearing mice treated with the other immunogens (P < 0.01). Likewise, Kaplan-Meier survival analysis further showed that tumor-challenged mice treated with the mHSP110-E711-20 complex exhibited the most markedly prolonged survival; however, the mHSP70-E711-20 and CFA-emulsified E711-20 immunizations also produced significant antitumor effects compared to the mHSP110 alone, mHSP70 alone, and E749-57 alone and the PBS group (Fig. 5B).

Fig 5.

Fig 5

Open in a new tab

Antitumor effects of the indicated immunogens on established tumors. (A) Data represent the mean tumor volumes of all 10 mice per group. *, P < 0.01 compared to the mHSP110, mHSP70, E711-20, or PBS groups; #, P < 0.01 compared to all other groups. (B) Time to death over a 70-day period is plotted as a Kaplan-Meier survival curve. Deaths include those of mice that were euthanized upon becoming moribund due to tumor burden.

DISCUSSION

The utility of HSPs as peptide adjuvants has been widely researched. The HSP family members HSP70, HSP90, GP96, and HSP110 have been successfully applied in the clinical setting as vaccine adjuvants targeting cancers and infections (39–41). Peptides can also be complexed with either tissue-derived or recombinant HSPs in vitro to generate HSP-peptide complexes for use as peptide-specific vaccines (22, 42, 43). However, clinical application of tissue-derived HSPs is limited by the requirement of a surgical specimen of sufficient quantity to purify the HSP. We had previously verified that mHSP110 significantly enhances the immunity of the H-2d-restricted HPV E749-57 epitope in a mouse model (29), but it remained unknown whether HSP110 enhances the immunogenicity of different major histocompatibility complex (MHC)-restricted epitope peptides. In the current study, we investigated the efficacy of the mHSP110 molecular chaperone as an immunoadjuvant to enhance the immunogenicity of the HLA-A2-restricted HPV16 E711-20 peptide vaccine. The results further proved the broad applicability of a peptide vaccine strategy based on the HSP110 chaperone.

We selected the HPV16 E711-20 epitope peptide as the target molecule because it had been previously verified as an HLA-A*0201-restricted CTL epitope of HPV16 E7. Furthermore, this epitope had been detected in patients with squamous cell carcinoma of the oropharynx (44), and an HPV16 E711-20-specific CTL cell line had been established (45). Several strategies based on the E711-20 peptide, including DNA vaccine format and single amino acid substitution, have shown improved immunogenicity in in vivo systems (38, 46). However, the human E711-20 epitope has been less extensively studied than the mouse H2-Db-restricted CTL epitope E749-57. Thus, further investigations on the E711-20 epitope are necessary, especially for improving the immunity of this epitope in transgenic animal models, which will have direct implications for applications in human HPV infection cases.

The CTL response is a key component of the immune response against tumor cells. Therefore, the focus of this study was to observe whether mHSP110 enhanced the efficient activation and expansion of functional CTLs specific to the HPV16 E711-20 peptide. Indeed, the results presented herein demonstrate that the mHSP110-E711-20 complex elicits epitope-specific splenocyte proliferation and E711-20-specific IFN-γ secretion. Importantly, the mHSP110-E711-20 complex immunization produced very strong cytolytic effects on target T2 cells pulsed with the E711-20 peptide, and even the HLA-A2 transgenic TC-1 cells that process and present the E711-20 epitope from the E7 protein in TC-1 cells. Besides the strong ex vivo antitumor immune response, the mHSP110-E711-20 complex can also reduce the tumor volume and enhance the survival of tumor-bearing mice.

The mHSP110-E711-20 complex showed higher antitumor effects than CFA-emulsified E711-20 peptide. This observation is in accordance with previous findings by Wang et al., which showed that the HSP110 chaperone gp100 generated a higher antitumor immune response than CFA-gp100 (28). In addition, we also found that the mHSP110-E711-20 complex is stronger than the mHSP70-E711-20 complex in priming the host antitumor response. This observation may reflect the fact that HSP110 has a much higher protein- and peptide-binding affinity than the HSP70 family (28, 47–49).

In conclusion, our data encourage further investigations of HSP110 as a promising candidate immunomodulator for peptide-based human cancer vaccines, such as the HLA-A2-restricted E711-20 epitope.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Natural Science Foundation of China (no. 30972800).

No competing financial interests exist.

Footnotes

Published ahead of print 8 May 2013

REFERENCES

  • 1. Adams M, Borysiewicz L, Fiander A, Man S, Jasani B, Navabi H, Lipetz C, Evans AS, Mason M. 2001. Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer. Vaccine 19:2549–2556 [DOI] [PubMed] [Google Scholar]
  • 2. Bernat A, Avvakumov N, Mymryk JS, Banks L. 2003. Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300. Oncogene 22:7871–7881 [DOI] [PubMed] [Google Scholar]
  • 3. Choo CK, Ling MT, Suen CK, Chan KW, Kwong YL. 2000. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells. Gynecol. Oncol. 78:293–301 [DOI] [PubMed] [Google Scholar]
  • 4. Zheng Y, Zhang Y, Ma Y, Wan J, Shi C, Huang L. 2008. Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region. J. Microbiol. 46:728–736 [DOI] [PubMed] [Google Scholar]
  • 5. Davidson JS, Demsey D. 2012. Atypical fibroxanthoma: clinicopathologic determinants for recurrence and implications for surgical management. J. Surg. Oncol. 105:559–562 [DOI] [PubMed] [Google Scholar]
  • 6. Liao JB, Jean S, Wilkinson-Ryan I, Ford AE, Tanyi JL, Hagemann AR, Lin LL, McGrath CM, Rubin SC. 2011. Vaginal intraepithelial neoplasia (VAIN) after radiation therapy for gynecologic malignancies: a clinically recalcitrant entity. Gynecol. Oncol. 120:108–112 [DOI] [PubMed] [Google Scholar]
  • 7. Liao SJ, Hu XJ, Han LF, Jiang XF, Xia X, Wang W, Lu YP, Wang SX, Ma D. 2009. Preparation of human papillomavirus 16 E7 peptide vaccine and its effectiveness in vitro and in vivo. Zhonghua Fu Chan Ke Za Zhi 44:903–908 (In Chinese) [PubMed] [Google Scholar]
  • 8. Liu DW, Yang YC, Lin HF, Lin MF, Cheng YW, Chu CC, Tsao YP, Chen SL. 2007. Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients. J. Virol. 81:2869–2879 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Schreurs MW, Scholten KB, Kueter EW, Ruizendaal JJ, Meijer CJ, Hooijberg E. 2003. In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones. J. Immunol. 171:2912–2921 [DOI] [PubMed] [Google Scholar]
  • 10. Feltkamp MC, Smits HL, Vierboom MP, Minnaar RP, de Jongh BM, Drijfhout JW, ter Schegget J, Melief CJ, Kast WM. 1993. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol. 23:2242–2249 [DOI] [PubMed] [Google Scholar]
  • 11. Brinkman JA, Hughes SH, Stone P, Caffrey AS, Muderspach LI, Roman LD, Weber JS, Kast WM. 2007. Therapeutic vaccination for HPV induced cervical cancers. Dis. Markers 23:337–352 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Kanodia S, Da Silva DM, Kast WM. 2008. Recent advances in strategies for immunotherapy of human papillomavirus-induced lesions. Int. J. Cancer 122:247–259 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Lazoura E, Apostolopoulos V. 2005. Insights into peptide-based vaccine design for cancer immunotherapy. Curr. Med. Chem. 12:1481–1494 [DOI] [PubMed] [Google Scholar]
  • 14. Scheibenbogen C, Letsch A, Schmittel A, Asemissen AM, Thiel E, Keilholz U. 2003. Rational peptide-based tumour vaccine development and T cell monitoring. Semin. Cancer Biol. 13:423–429 [DOI] [PubMed] [Google Scholar]
  • 15. Lazoura E, Apostolopoulos V. 2005. Rational peptide-based vaccine design for cancer immunotherapeutic applications. Curr. Med. Chem. 12:629–639 [DOI] [PubMed] [Google Scholar]
  • 16. Meng WS, Butterfield LH. 2002. Rational design of peptide-based tumor vaccines. Pharm. Res. 19:926–932 [DOI] [PubMed] [Google Scholar]
  • 17. Ohno S, Kohyama S, Taneichi M, Moriya O, Hayashi H, Oda H, Mori M, Kobayashi A, Akatsuka T, Uchida T, Matsui M. 2009. Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice. Vaccine 27:3912–3920 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Behr TM, Gotthardt M, Barth A, Behe M. 2001. Imaging tumors with peptide-based radioligands. Q. J. Nucl. Med. 45:189–200 [PubMed] [Google Scholar]
  • 19. Montomoli E, Piccirella S, Khadang B, Mennitto E, Camerini R, De Rosa A. 2011. Current adjuvants and new perspectives in vaccine formulation. Expert Rev. Vaccines 10:1053–1061 [DOI] [PubMed] [Google Scholar]
  • 20. Gauthier A, Martin-Escudero V, Moore L, Ferko N, de Sanjose S, Perez-Escolano I, Catala-Lopez F, Ferrer E, Bosch FX. 2008. Long-term clinical impact of introducing a human papillomavirus 16/18 AS04 adjuvant cervical cancer vaccine in Spain. Eur. J. Public Health 18:674–680 [DOI] [PubMed] [Google Scholar]
  • 21. Pedersen C, Petaja T, Strauss G, Rumke HC, Poder A, Richardus JH, Spiessens B, Descamps D, Hardt K, Lehtinen M, Dubin G; HPV Vaccine Adolescent Study Investigators Network 2007. Immunization of early adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant. J. Adolesc. Health 40:564–571 [DOI] [PubMed] [Google Scholar]
  • 22. Wang XY, Manjili MH, Park J, Chen X, Repasky E, Subjeck JR. 2004. Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins. Methods 32:13–20 [DOI] [PubMed] [Google Scholar]
  • 23. Goeckeler JL, Petruso AP, Aguirre J, Clement CC, Chiosis G, Brodsky JL. 2008. The yeast Hsp110, Sse1p, exhibits high-affinity peptide binding. FEBS Lett. 582:2393–2396 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Easton DP, Kaneko Y, Subjeck JR. 2000. The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s. Cell Stress Chaperones 5:276–290 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Kim HL, Sun X, Subjeck JR, Wang XY. 2007. Evaluation of renal cell carcinoma vaccines targeting carbonic anhydrase IX using heat shock protein 110. Cancer Immunol. Immunother. 56:1097–1105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Manjili MH, Henderson R, Wang XY, Chen X, Li Y, Repasky E, Kazim L, Subjeck JR. 2002. Development of a recombinant HSP110-HER-2/neu vaccine using the chaperoning properties of HSP110. Cancer Res. 62:1737–1742 [PubMed] [Google Scholar]
  • 27. Manjili MH, Wang XY, Chen X, Martin T, Repasky EA, Henderson R, Subjeck JR. 2003. HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice. J. Immunol. 171:4054–4061 [DOI] [PubMed] [Google Scholar]
  • 28. Wang XY, Chen X, Manjili MH, Repasky E, Henderson R, Subjeck JR. 2003. Targeted immunotherapy using reconstituted chaperone complexes of heat shock protein 110 and melanoma-associated antigen gp100. Cancer Res. 63:2553–2560 [PubMed] [Google Scholar]
  • 29. Ren F, Xu Y, Mao L, Ou R, Ding Z, Zhang X, Tang J, Li B, Jia Z, Tian Z, Ni B, Wu Y. 2010. Heat shock protein 110 improves the antitumor effects of the cytotoxic T lymphocyte epitope E7(49–57) in mice. Cancer Biol. Ther. 9:134–141 [DOI] [PubMed] [Google Scholar]
  • 30. Kast WM, Brandt RM, Drijfhout JW, Melief CJ. 1993. Human leukocyte antigen-A2.1 restricted candidate cytotoxic T lymphocyte epitopes of human papillomavirus type 16 E6 and E7 proteins identified by using the processing-defective human cell line T2. J. Immunother. Emphasis Tumor Immunol. 14:115–120 [DOI] [PubMed] [Google Scholar]
  • 31. Liu WJ, Liu XS, Zhao KN, Leggatt GR, Frazer IH. 2000. Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes. Virology 273:374–382 [DOI] [PubMed] [Google Scholar]
  • 32. Ressing ME, Sette A, Brandt RM, Ruppert J, Wentworth PA, Hartman M, Oseroff C, Grey HM, Melief CJ, Kast WM. 1995. Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides. J. Immunol. 154:5934–5943 [PubMed] [Google Scholar]
  • 33. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T. 1992. Allele and haplotype frequencies for HLA and complement loci in various ethnic groups, p 1065–1220 In Tsuji K, Aizawa M, Sasazuki T. (ed), HLA 1991: proceedings of the Eleventh International Histocompatibility Workshop and Conference, Oxford University Press, Oxford, United Kingdom [Google Scholar]
  • 34. Liang B, Zhu L, Liang Z, Weng X, Lu X, Zhang C, Li H, Wu X. 2006. A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China. Cell. Mol. Immunol. 3:453–458 [PubMed] [Google Scholar]
  • 35. He Y, Mao L, Lin Z, Deng Y, Tang Y, Jiang M, Li W, Jia Z, Wang J, Ni B, Wu Y. 2008. Identification of a common HLA-A*0201-restricted epitope among SSX family members by mimicking altered peptide ligands strategy. Mol. Immunol. 45:2455–2464 [DOI] [PubMed] [Google Scholar]
  • 36. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, Wu TC. 1996. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 56:21–26 [PubMed] [Google Scholar]
  • 37. Nanjundappa RH, Wang R, Xie Y, Umeshappa CS, Xiang J. 2012. Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice. Vaccine 30:3519–3525 [DOI] [PubMed] [Google Scholar]
  • 38. Peng S, Trimble C, He L, Tsai YC, Lin CT, Boyd DA, Pardoll D, Hung CF, Wu TC. 2006. Characterization of HLA-A2-restricted HPV-16 E7-specific CD8(+) T-cell immune responses induced by DNA vaccines in HLA-A2 transgenic mice. Gene Ther. 13:67–77 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Murshid A, Gong Calderwood JSK. 2008. Heat-shock proteins in cancer vaccines: agents of antigen cross-presentation. Expert Rev. Vaccines 7:1019–1030 [DOI] [PubMed] [Google Scholar]
  • 40. Segal BH, Wang XY, Dennis CG, Youn R, Repasky EA, Manjili MH, Subjeck JR. 2006. Heat shock proteins as vaccine adjuvants in infections and cancer. Drug Discov. Today 11:534–540 [DOI] [PubMed] [Google Scholar]
  • 41. Srivastava PK, Callahan MK, Mauri MM. 2009. Treating human cancers with heat shock protein-peptide complexes: the road ahead. Expert Opin. Biol. Ther. 9:179–186 [DOI] [PubMed] [Google Scholar]
  • 42. Heike M, Noll B, Meyer zum Buschenfelde KH. 1996. Heat shock protein-peptide complexes for use in vaccines. J. Leukoc. Biol. 60:153–158 [DOI] [PubMed] [Google Scholar]
  • 43. Li Z. 1997. Priming of T cells by heat shock protein-peptide complexes as the basis of tumor vaccines. Semin. Immunol. 9:315–322 [DOI] [PubMed] [Google Scholar]
  • 44. Hoffmann TK, Arsov C, Schirlau K, Bas M, Friebe-Hoffmann U, Klussmann JP, Scheckenbach K, Balz V, Bier H, Whiteside TL. 2006. T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx. Int. J. Cancer 118:1984–1991 [DOI] [PubMed] [Google Scholar]
  • 45. Youde SJ, McCarthy CM, Thomas KJ, Smith KL, Man S. 2005. Cross-typic specificity and immunotherapeutic potential of a human HPV16 E7-specific CTL line. Int. J. Cancer 114:606–612 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Schreurs MW, Kueter EW, Scholten KB, Lemonnier FA, Meijer CJ, Hooijberg E. 2005. A single amino acid substitution improves the in vivo immunogenicity of the HPV16 oncoprotein E7(11–20) cytotoxic T lymphocyte epitope. Vaccine 23:4005–4010 [DOI] [PubMed] [Google Scholar]
  • 47. Oh HJ, Easton D, Murawski M, Kaneko Y, Subjeck JR. 1999. The chaperoning activity of hsp110. Identification of functional domains by use of targeted deletions. J. Biol. Chem. 274:15712–15718 [DOI] [PubMed] [Google Scholar]
  • 48. Wang XY, Kazim L, Repasky EA, Subjeck JR. 2001. Characterization of heat shock protein 110 and glucose-regulated protein 170 as cancer vaccines and the effect of fever-range hyperthermia on vaccine activity. J. Immunol. 166:490–497 [DOI] [PubMed] [Google Scholar]
  • 49. Oh HJ, Chen X, Subjeck JR. 1997. Hsp110 protects heat-denatured proteins and confers cellular thermoresistance. J. Biol. Chem. 272:31636–31640 [DOI] [PubMed] [Google Scholar]

Articles from Clinical and Vaccine Immunology : CVI are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES