Dengue

Dengue Bulletin Volume 28 (2004)

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Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus – Derived, Modified Epitope Peptide, Using Dendritic Cells as a Peptide Delivery System

Yoshiki Fujii*, Hideyuki Masaki**^#, Takanori Tomura**, Kiyohiro Irimajiri*
and Ichiro Kurane***

*Department of Pharmacotherapy, Kinki University School of Pharmaceutical Sciences,
Higashi-Osaka, Osaka, Japan
** First Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama,
Osaka 589-8511, Japan
***Department of Virology 1, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan

Introduction

Major histocompatibility complex (MHC) class I – restricted, CD8+ cytotoxic T lymphocytes (CTLs) are known to play an essential role in the recovery from viral infection by lysing virus-infected cells[1]. There are two major strategies to induce CTL-mediated protective immunity. One is to  have CTL epitope expressed in host cells by infection with live viruses, or by administration of an expression plasmid vector (i.e. DNA vaccine) in which the epitope gene is incorporated. The other is immunization with a defined CTL epitope peptide. The former strategy is more physiological; however, the preparation of immunogen is often difficult and there is a potential risk that the immunogen may be pathogenic. Immunization with a CTL epitope peptide is relatively easy; however, the epitope varies depending on T cell receptor repertoires and MHC class I haplotypes. Furthermore, the peptide administrated into the body may be degraded or washed away soon. Thus, the peptide is usually less immunogenic for CTL induction, and an appropriate delivery system is necessary for induction of CTLs[2,3].

Dendritic cells (DCs), which are potent antigen-presenting cells, are postulated to be one of the peptide delivery systems for inducing CTLs[2,4]. It has been reported that intravenous immunization with DCs pulsed with virus-derived peptides, or tumour-derived peptides, elicits specific CTLs[5-7].

Dengue viruses cause dengue fever and dengue haemorrahgic fever/dengue shock syndrome (DHF/DSS). Vaccine development against dengue virus infection has not been accomplished yet. It is important to analyse CTL responses elicited by a single epitope in order to understand the role of CTLs in dengue virus infection. In the present study, we employed a single 9-amino acid (a.a.) peptide, in which C-terminal residue was replaced to provide the complete H-2Kd-binding motif. This peptide was a derivative of the defined H-2Kd-restricted 9-a.a. CTL epitope peptide that corresponds to the residues 298-306 of NS3 of dengue virus types 2 and 4[8]. We examined whether intravenous immunization with bone marrow-derived DCs pulsed with this peptide elicited specific CTL response.

Materials and methods

Mice

Female BALB/cAJcl mice were purchased from Clea, Japan, and were maintained in the Animal Facility of Kinki University School of Medicine under conventional conditions. Mice were used at the age of 6 to 12 weeks.

Cells

Murine mastcytoma line, P815 (H-2d), was used as target cells in CTL assays. The cells were maintained in RPMI 1640 medium (Sigma, St. Louis, MO) with 5x10-5M 2-mercaptoethanol (2-ME), 100U penicillin, 100g/ml streptomycin, 10mM HEPES, and 10% heat-inactivated fetal calf serum (Complete medium) at 37 °C in 5% CO2.

Peptides

The peptide-1 (GYISTRVEM), which corresponds to the amino acid residues 298-306 of NS3 of dengue virus types 2 and 4, and the peptide-2 (GYISTRVEL) were synthesized with 9-fluorenylmethoxycarbonyl chemistry by Sigma Genosis, Japan. The purity was determined to be 95.0% for the peptide-1 and 96.2% for the peptide-2 by reverse phase HPLC.

Induction of dendritic cells

BALB/c mouse bone marrow cells (9x105) were cultured in 1ml of AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 20ng/ml mouse GM-CSF (R&D Systems, Minneapolis, MN) in 24-well plate at 37 °C in 5% CO2. On days 4 and 6, 50 to 75% volume of the culture medium was changed with the fresh one supplemented with the same amount of GM-CSF. On day 7, cells were harvested, subjected to flow cytometry analysis and used as DCs for immunization.

Flow cytometry analysis

Bone marrow-derived cells (4x105) were incubated with 1g of FITC-conjugated anti-mouse I-Ad/I-Ed antibody (BD PharMingen, San Diego, CA), PE-conjugated anti-mouse CD86 antibody (BD PharMingen), and biotinylated anti-mouse CD11c (BD PharMingen) in 100l of phosphate buffered saline (PBS) containing 0.02% NaN3 (PBS/NaN3) at 4 °C for 20 minutes. Same amounts of FITC-conjugated rat IgG2a, (BD PharMingen), PE-conjugated rat IgG2a, (BD PharMingen) and biotinylated hamster IgG group 1,  (BD PharMingen) were used as isotype controls. The cells were washed three times with PBS/NaN3 at 4 °C, and then incubated with 0.1g of streptoavidin-conjugated Cy-ChromeTM (BD PharMingen) in 100l of PBS/NaN3 at 4 °C for 20 minutes. The cells were washed three times, fixed with 1ml of PBS containing 1% paraformaldehyde, and analysed by a FACS Calibur (Becton Dickinson, San Jose, CA) and CELL QuestTM version 3.3 software.

Immunization and CTL induction

Bone marrow cells (10x106) stimulated with GM-CSF for 7 days were incubated in the presence of 10M peptide-2 in 1ml of AIM-V at 37 °C for 2 hours. The cells were washed two times with RPMI-1640. Peptide-2-pulsed cells (2x106) were injected intravenously into BALB/c mice. Four weeks later, the spleens were collected, minced into single cell suspension, erythrocyte-lysed, and treated with anti-CD4 antibody (BD PharMingen) at the rate of 1g/1x107 cells and 10% baby rabbit complement (Cederlane, Hornby, Ont, Canada) to deplete CD4-positive cells. Five million cells were then co-cultured with the same number of peptide-2-pulsed, 33Gy X-ray-irradiated syngeneic spleen cells in 2ml of EHAA medium (Sigma) supplemented with 100g/ml nucleic acid precursors, 2mM L-glutamine, 5x10-5M 2-ME, 100U penicillin, 100g/ml streptomycin, 10mM HEPES, and 10% fetal calf serum in 24-well plate at 37 °C in 5% CO2. On day 4, half volume of the medium was replaced with fresh one, and 10 U recombinant mouse IL-2 was added. On day 7, the cells were harvested and used as CTL (cytotoxic T lymphocyte) effector cells. Mice were also immunized by subcutaneous injection with 1 n mole of the peptide emulsified with complete Freund adjuvant (CFA) into two-foot pads. In this immunization protocol, draining lymph node (popliteal lymph nodes) cells were used as the effector cells after stimulation in vitro with the peptide as described above. When mice were immunized with the peptide-1, the cells were stimulated in vitro with peptide-1-pulsed spleen cells. 

Cytotoxicity assays

P815 cells (1x106) were pulsed with the peptide at a concentration of 10M in complete medium at 37 °C for 3 hours. The cells were labelled with 100 Ci of Na251CrO4 (NEN Life Science Products, Boston, MA) for one hour, then washed three times and suspended in complete medium. Peptide-pulsed, 51Cr-labelled cells were seeded in 96-well V-bottom plate at 1.5x103 cells in 100l of complete medium per well. Effector cells were added to the plate to make various effector/target ratios (E/T ratios) in a total volume of 0.2ml per well, and the plate was incubated at 37 °C in 5% CO2 for four hours. The supernatant fluids were harvested with a Supernatant Collecting System (Skatoron, Lier, Norway), and 51Cr content was measured by a gamma counter (Aloka model ARC-300). Maximum 51Cr release was determined by adding 0.1% Triton X, and spontaneous 51Cr release was determined with the wells that contained target cells and medium only. Assays were performed in triplicate, and the mean value was used to calculate percent-specific lysis with the following formula: % specific lysis = 100 x [(release with effector cells – spontaneous release) / (maximum release – spontaneous release)]. Spontaneous release did not exceed 28.9% of the maximum release.

Results and discussion

It is known that mature murine DCs strongly express major histocompatibility complex (MHC) class II antigen, co-stimulatory molecules CD80 and CD86, and CD11c, the  chain of p150/95 2-integrin[9,10]. We examined the expression of MHC class II antigen I-Ad/I-Ed, CD86, and CD11c on BALB/c mouse bone marrow cells, which were cultured with GM-CSF for seven days, by flow cytometry three colour analysis, and evaluated the purity of DC. As shown in Figure 1a, 25.07% of the bone marrow cells strongly expressed both I-Ad/I-Ed and CD86. The percentage of CD11c-positive cells in these double positive cells was 95.81% (Figures 1b and 1c). In contrast, freshly isolated bone marrow cells did not express these surface molecules (data not shown). These results suggest that bone marrow cells were differentiated into DCs during the culture with GM-CSF for seven days and that DCs accounted for one-fourth of the entire population.

Figure 1. Expression of the dendritic cell markers on bone marrow cells after culture in the presence of GM-CSF for 7 days

[BALB/c mouse bone marrow cells were cultured for 7 days in AIM-V medium with 20 ng/ml of murine GM-CSF. The cells were stained with anti – I-A^d/I-E^d – FITC, anti-CD86 – PE, anti-CD11c – biotin, and streptoavidin – Cy-Chrome^TM. (a); The cells in the region (R1) strongly expressed I-A^d/I-E^d and CD86, and accounted for 25.07% of the entire cultured bone marrow cells. (b) & (c): The cells in the region (R1) were examined for the expression of CD11c. 95.81% expressed CD11c.]

We first attempted to induce specific CTLs by two foot pad immunizations with the peptide-1 (GYISTRVEM) emulsified with CFA. Specific CTL activity was not detected in draining lymph node cells. (Table, Experiment no. 1). We speculated that the inability of the peptide-1 to induce specific CTLs might partly be due to the low binding affinity to H-2K^d MHC class I molecule, because the peptide-1 possesses only one anchor residue (Y) for binding to H-2K^d molecule. We, therefore, prepared the peptide-2 (GYISTRVEL) in which the last residue M of the peptide-1 was substitutedfor L in order to provide the complete H-2K^d-binding motif^[11].

It was reported that immunization with virus epitope peptide–pulsed DCs efficiently induced virus-specific CD8⁺ CTLs and protective immunity^[5,6]. We attempted to induce specific CTLs by immunization with peptide-2-pulsed DCs. We intravenously injected 2x10⁶ bone marrow cells, which were stimulated with GM-CSF for seven days and pulsed with the peptide-2, into BALB/c mice. One-fourth of the cultured bone marrow cells were DCs and it was reported that intravenous immunization with 1x10⁵ to 5x10⁵ purified DCs pulsed with peptides induced antiviral immunity^[5]. Spleen cells from the mice immunized with peptide-2-pulsed DCs lysed peptide-2-pulsed P815 cells in a dose dependent fashion after stimulation in vitro with X-ray-irradiated, peptide-2-pulsed syngeneic spleen cells in the presence of recombinant IL-2 for seven days (Table, Experiment no. 2). This result demonstrates that intravenous immunization with peptide-2-pulsed DCs induced peptide-2-specific CTLs.

Table. Induction of specific CTLs by immunization with peptide-2-pulsed dendritic cells and the peptide-2 emulsified with complete Freund adjuvant

* ⁵¹Cr–labeled P815 mastcytoma (H-2^d) pulsed with the peptide (10µM, 3hours) were used as target cells.

Specific CTL activity was observed in the draining lymph node cells after immunization with peptide-2/CFA. Interestingly, CTLs induced by peptide-2/CFA demonstrated lower levels of non-specific cytotoxic activity to P815 cells than those induced by peptide-2-pulsed DCs. Peptide-2/CFA-induced CTLs lysed peptide-1-pulsed target cells as well as peptide-2-pulsed ones. (Table, Experiment nos. 2 and 3). These results suggest that the peptide-2 which has the complete binding motif to H-2k^d molecule can induce specific CTLs when used with CFA, and that peptide-2-specific CTLs also lyse original peptide-1-pulsed target cells. Induction of higher levels of non-specific cytotoxicity by immunization with DCs may be due to the high antigen presentation ability of DCs to prime various repertoires of T cells. The other possibility is the difference in the source of lymphocytes. We observed that spleen-derived lymphocytes tended to show higher levels of non-specific lysis than lymph node-derived cells (data not shown). It seems that the draining lymph node cells from mice immunized with the peptide and CFA are a better source of CTLs than the spleen cells from those immunized with peptide-pulsed DCs because of low non-specific cytotoxic activiity. The data, however, only suggest how efficiently measurable specific CTLs can be induced in vitro. It is plausible that intravenous immunization with peptide-pulsed DCs induces higher levels of specific CTLs in vivo and protective immunity against viral infections. Moreover, CFA is not accepted for human use. Thus, immunization strategy using peptide-pulsed DCs is still worth investigating for induction of CTL-mediated anti-dengue virus immunity.

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