Institute of Immunology, Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Straße 28, D-72076 Tübingen, Germany1
EMC microcollections GmbH, D-72070 Tübingen, Germany2
Institut für Organische Chemie, Universität Tübingen, D-72076 Tübingen, Germany3
Author for correspondence: Armin Saalmüller. Fax +49 7071 967303. e-mail armin.saalmueller{at}tue.bfav.de
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Abstract |
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Introduction |
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Outbreaks of this disease occur frequently in several countries, usually with severe economic consequences (Edwards et al., 2000 ; Stegeman et al., 2000
). Vaccination based on lapinized C strains of CSFV and temperature-sensitive mutants are being used in many parts of the world (Aynaud, 1988
). However, these kinds of vaccines are prohibited from use within the European Union and several other countries due to the impossibility of differentiating between vaccinated and infected animals. In order to overcome the disadvantages of conventional vaccines, recombinant proteins (van Rijn et al., 1996
), virus vector vaccines (Rümenapf et al., 1991
; van Zijl et al., 1991
; Hooft van Iddekinge et al., 1996
), DNA vaccines (Andrew et al., 2000
) and peptide vaccines (DiMarchi et al., 1986
) have been proposed.
Synthetic peptides are promising candidate vaccines for the control of virus diseases. Peptide vaccines based on epitopes have been shown to induce a specific immune response (Deres et al., 1989 ) and to protect the host against the disease (Bittle et al., 1982
; Menne et al., 1997
; Wiesmüller et al., 1989
). Thus, for the development of a synthetic peptide vaccine, characterization of the porcine immune response against CSFV is necessary. Studies on the cellular immune response to CSFV have been described previously (Kimman et al., 1993
). Viral proteins responsible for the induction of the virus-specific T-cell response were examined and a cytotoxic T-lymphocyte (CTL) epitope from the NS4A protein was identified (Pauly et al., 1995
). Also, the knowledge of allele-specific peptide motifs of major histocompatibility complex (MHC) class I (Falk et al., 1991
) and class II (Falk et al., 1994
) molecules is of great importance for the prediction and identification of species-specific T-cell epitopes. The use of synthetic, overlapping peptides that span the regions encoding the viral proteins is one of the strategies for the identification of T-cell epitopes (Muller et al., 1996
; Blanco et al., 2001
; Ober et al., 1998
; Menne et al., 1997
). The aim of this study was to characterize the T-cell response to CSFV and to identify new CSFV T-cell epitopes using the cellular immune response of the natural host. A T-cell epitope mapping was performed with a set of 573 CSFV-derived, overlapping peptides spanning 82% of the single ORF of the genome. Further experiments showed that a peptide derived from the NS23 protein contained a helper T-cell epitope as well as a CTL epitope in its sequence.
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Methods |
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Monoclonal antibodies (mAb).
Murine mAbs against porcine MHC class I (mAb 74-11-10, mouse IgG2b) (Pescovitz et al., 1984 ), MHC class II (mAb MSA3, mouse IgG2a) (Hammerberg & Schurig, 1986
) and CD4 (mAb 74-12-4, mouse IgG2b) (Pescovitz et al., 1984
) were kindly provided by J. K. Lunney (USDA, Agricultural Research Service, Beltsville, MD, USA). The porcine CD8 mAb (mAb 11/295/33, mouse IgG2a) (Jonjic & Koszinowski, 1984
) and mAb a18, a mouse IgG2a directed against glycoprotein E2 of CSFV (Weiland et al., 1990
), were established at the Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany.
Cells and viruses.
The Max cell line (Pauly et al., 1995 ) used as target cells for the cytotoxicity assays was established at the Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany. The cell line was grown in Dulbeccos modified Eagles medium (DMEM) (Gibco BRL) supplemented with 10% foetal calf serum (FCS), 2 mM L-glutamine, 0·05 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.2), 100 IU/ml penicillin and 0·1 mg/ml streptomycin sulphate. CSFV-Alfort/187 (Ruggli et al., 1996
) and CSFV-Glentorf (Pittler et al., 1968
) were a gift from R. Ahl, Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany. Both virus strains were propagated in the STE cell line (McClurkin & Norman, 1966
) kindly provided by R. Ahl. Virus titres were determined by an indirect peroxidase-linked antibody assay using mAb a18 (Weiland et al., 1990
; Kosmidou et al., 1995
). The STE cell line was grown in 40% MEMNEAA (Gibco BRL), 40% Leibovitzs L-15 medium (Gibco BRL), 10% tryptose phosphate broth, 10% horse serum, 100 IU/ml penicillin and 0·1 mg/ml streptomycin sulphate. All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Synthesis and analysis of synthetic peptides.
A collection of 573 peptides spanning 82% of the amino acid sequence of the CSFV-Glentorf protein coding regions were synthesized using solid-phase multiple peptide synthesis (Jung & Beck-Sickinger, 1992 ) on a fully automated synthesizer (Syro). The 15-mer peptides, which overlapped each other by 10 residues, were prepared by solid-phase peptide synthesis using Fmoc/tBu chemistry. Syntheses were carried out on p-benzyloxybenzyl alcohol resin loaded with the first amino acid. The resin was distributed in 2035 mg aliquots (15 µmol) to filter tubes positioned in a valve block. Fmoc deprotections were carried out with 50% piperidine in dimethylformamide (DMF). Washing steps were done with DMF. Double couplings (1 h each) were performed with Fmoc amino acids in 10-fold excess and 1-hydroxybenzotriazole/diisopropylcarbodiimide activation in DMF. Coupling reagents were filtered off and the resins were washed with DMF. Peptides were cleaved from the resins and side chain-deprotected with trifluoroacetic acid:phenol:ethanedithiol:thioanisole:water (82·5:5:2·5:5:5) for 3 h. Products were filtered from the resins and precipitated at -20 °C by the addition of diethylether. Precipitates were washed twice by sonication in diethylether and lyophilized from water:tert-butyl alcohol (1:4). The identity of each peptide was confirmed by electrospray mass spectrometry (triple-quadrupol, Micromass) and most of the peptides were greater than 70% pure, as determined by reverse-phase high-pressure liquid chromatography (Gynkotek) at 214 nm. All peptides (1 mg/ml) were dissolved in 1% DMSO in water as a stock solution (stored at -20 °C) and further diluted in culture medium.
Isolation of PBMCs.
Infected pigs were bled every 1 or 2 weeks after the first infection. PBMCs were separated using density gradient centrifugation, as described previously (Summerfield et al., 1996 ). Cells were frozen and stored in liquid nitrogen until use.
Lymphoproliferation assays.
For the detection of CSFV-specific proliferation, PBMCs (105 cells per well) derived from CSFV-infected pigs were cultivated in MEM -medium supplemented with 10% FCS, 2 mM L-glutamine, 0·05 mM 2-mercaptoethanol, 10 mM HEPES, 100 IU/ml penicillin and 0·1 mg/ml streptomycin sulphate as quadruplicates in 96-well round-bottom microplates (Greiner).
Cells were stimulated either with virus (104 TCID50 CSFV-Alfort/187 or 5x104 TCID50 CSFV-Glentorf per well) or with synthetic peptides (12·5 and 25 µg/ml). After incubation for 5 days at 37 °C in 5% CO2, proliferative responses were determined by adding [methyl-3H]thymidine for 18 h to the cultures (1 µCi per well) (Amersham). Cells were collected using a cell harvester system (Inotech). Incorporation of [3H]thymidine was measured in a Microbeta 1450 -counter (Wallac). Results were expressed as stimulation indexes (SI), defined as the mean c.p.m. of stimulated cultures/mean c.p.m. of control cultures. The response was considered to be positive when the SI was
2.
Inhibition of CSFV-derived peptide-specific proliferation.
For the determination of MHC restriction of the presentation of peptide 290, mAbs directed against the porcine CD4 (mAb 74-12-4), CD8 (mAb 11/295/33), MHC class I (mAb 74-11-10) and MHC class II (mAb MSA3) molecules were added to the cultures to test their capacity to inhibit peptide-specific proliferation of CSFV-primed PBMCs. Thus, PBMCs were incubated with the appropriate dilution of mAb (hybridoma supernatant anti-CD4, anti-CD8, anti-MHC class I or anti-MHC class II) for 2 h before use in proliferation assays, as described previously (Summerfield et al., 1996 ).
Cytotoxicity assays.
A chromium-release assay was used to measure CTL responses. CSFV-specific CTLs were generated by in vitro restimulation with virus (5x104 TCID50 CSFV-Glentorf per well) for 6 days in 96-well round-bottom microplates.
MAX cells infected with CSFV-Glentorf at an m.o.i. of 1 for 48 h were used as target cells in cytotoxicity assays. Then, 1x106 target cells were labelled with 100 µCi Na251CrO4 (Amersham) for 1 h at 37 °C in 5% CO2. After washing, cells were resuspended in cell culture medium. For CTL experiments with peptides, 4x104 non-infected chromium-labelled target cells were incubated at 37 ° C and 5% CO2 with 20 µg of the respective peptides for 1 h. CSFV-infected or peptide-loaded target cells (1x103 cells per well) were added to various concentrations of effector cells (ranging from 1·25x104 to 5x104 cells per well). All experiments were performed in triplicate cultures. Cells were centrifuged at 100 g for 5 min and incubated for 4 h at 37 °C in 5% CO2. After pelleting the cells at 600 g for 10 min, the chromium levels in the supernatants were measured using a Cobra Autogamma -counter (Packard). The percentage of specific cytolytic activity was calculated as follows: [(c.p.m. experimental release)-(c.p.m. spontaneous release)/(c.p.m. total release)-(c.p.m. spontaneous release)]x100. Mock-infected or negative peptide-loaded target cells served as the controls.
Interferon (IFN)-
ELISPOT assay.
The ELISPOT assay was used to enumerate CSFV- and peptide-specific IFN--secreting cells. For this, 96-well nitrocellulose plates (Millipore) were coated with 100 µl per well of mouse anti-pig IFN-
mAb (5 µg/ml) (Endogen) diluted in PBS. After overnight incubation at 4 °C, wells were washed with PBS and incubated with 200 µl of MEM
-medium for 1 h at room temperature. Different concentrations of PBMCs from CSFV-infected pigs (ranging from 1·25x105 to 1x106 cells per well) were added in duplicate (each 100 µl) to the antibody-coated plates and incubated with 100 µl CSFV (2x104 TCID50 CSFV-Alfort/187 or 5x104 TCID50 CSFV-Glentorf per well) for 48 h. In peptide-sensitization experiments, 1x106 PBMCs per well (100 µl) were incubated with 100 µl of various concentrations of peptides (ranging from 5 to 100 µg/ml). PBMCs incubated with supernatant of mock-infected cells or irrelevant peptide 5 served as the negative controls.
After incubation at 37 °C in 5% CO2 for 48 h, wells were washed with PBS and incubated with 100 µl of rabbit anti-pig IFN- polyclonal antibody (2·5 µg/ml) (Endogen) for 1 h at room temperature.
After washing with PBS, 100 µl per well of goat anti-rabbit peroxidase-conjugated IgG (Dianova) at a 1:200 dilution in PBS was added. After an additional 1 h of incubation at room temperature, wells were washed with PBS. For the development of IFN--specific spots, 3,3'-diaminobenzidine (Sigma) was added for 47 min at room temperature. The colorimetric reaction was stopped by washing the plates with PBS and then water. After drying at room temperature, coloured spots were counted using a stereomicroscope at a 40-fold magnification. Analyses were repeated three times; the SE between these repeats was less than 10%.
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Results |
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Secretion of IFN-
To confirm the reactivity of CSFV-specific T-cells against CSFV antigens, porcine T-cells were tested for their ability to produce IFN- upon virus as well as peptide stimulation in ELISPOT assays. These assays enable the detection of IFN-
release on a single cell level and a determination of T-cell frequencies. First, the number of cells releasing IFN-
was detected by spot formation in the presence of different strains of CSFV (Fig. 3a
, c
). Various concentrations of PBMCs (ranging from 1·25x105 to 1x106 cells per well) from CSFV-infected swine were tested. The mean number of spots increased with an increasing amount of cells. For spot detecting, 1x106 cells per well were considered to be appropriate. The release of IFN-
was CSFV-specific, as PBMCs incubated with supernatant from mock-infected cells presented only a few spots. High numbers of IFN-
-secreting cells were observed when PBMCs from infected pigs were stimulated with peptide 290 (Fig. 3b
, d
). The number of cells releasing IFN-
in response to peptide 290 was higher with PBMCs from swine infected with CSFV-Glentorf compared with the PBMCs from swine infected with CSFV-Alfort/187. The release of IFN-
by peptide 290 was shown to be sequence-specific, as only a few spots were observed after incubation of PBMCs with an irrelevant peptide. These results are an additional indication for an immunodominant role of peptide 290.
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Discussion |
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Of the 26 stimulating peptides derived from CSFV-Glentorf proteins, 18 induced a proliferative response in lymphocytes from a CSFV-Alfort/187-infected pig.
Of interest is the finding that the MHC d/d haplotype recognized common T-cell epitopes in both inbred swine. Otherwise, some peptides (e.g. peptides 133 and 134) with an identical amino acid sequence between both strains (Glentorf and Alfort/187) were recognized as T-cell epitopes only with PBMCs from one pig. This MHC-independent preference for epitopes may have been caused either by host factors or by specific virus factors. This point might be the content of further studies.
Five of the common T-cell epitopes (peptides 140, 148,162, 200 and 201) were located on the E2 protein. The E2 glycoprotein is the most antigenic glycoprotein of CSFV and an immune response against E2 alone was reported to be sufficient for protection against CSFV (van Zijl et al., 1991 ; Hulst et al., 1993
; König et al., 1995
). Our findings confirmed the striking antigenicity of this viral protein to induce an immune response. The stimulating peptides 200 and 201 derived from the E2 region are part of a B-cell epitope described previously (Yu et al., 1996
). The overlap of B- and T-cell epitopes in the same protein region has been described for other viral antigens (Rodriguez et al., 1994
). Peptide vaccines encompassing B- and T-cell epitopes can result in the enhancement of antibody responses (Borrás-Cuesta et al., 1987
; Collen et al., 1991
).
The pentadecapeptide 290, which is derived from NS23, showed the highest SI in all proliferation assays performed. The reactivity of PBMCs against this peptide was thus characterized further. To analyse the virus- and peptide-inducible production of IFN- from T-cells on a single cell level, ELISPOT assays were carried out. Advantages of the ELISPOT assay include sensitivity and efficiency in the detection of antigen-specific T-cells at the single cell level (Tanguay & Killion, 1994
). However, T-cell responses detected with this assay are limited to the T-cells that secrete IFN-
. This assay represents a useful tool for the ex-vivo determination of frequencies of antigen-specific T-cells. It is known that CD4+ T-cells (Th1) as well as CD8+ T-cells are able to produce IFN-
. In this study, virus-specific (CSFV-Glentorf and -Alfort/187) and peptide-specific (peptide 290) IFN-
release were detected by ELISPOT assay.
In order to characterize peptide 290-specific T-cells, the MHC restriction of the proliferative response to this peptide was determined. Proliferation in the presence of peptide 290 was inhibited principally by anti-MHC class II and anti-CD4, confirming an MHC class II restriction. However, a partial inhibition of proliferation of PBMCs was also observed with anti-MHC class I and anti-CD8 antibodies.
This MHC class I restriction of the pentadecapeptide 290 was tested in CTL assays for the capacity of the peptide to elicit cytotoxic T-cell responses. Virus-specific CTLs were tested against MAX cells loaded with peptide 290. The results indicated the presence of a CTL epitope in the sequence of peptide 290. Thus, peptide 290 has the capacity to prime both MHC class I and class II-restricted T-cells. The sequence encoded a helper T-cell and a CTL epitope, representing a multideterminant peptide. This protein region spanning amino acids 14461460 (peptide 290) is highly conserved among the different CSFV strains.
Lytic activities of CTLs against target cells infected with CSFV were higher than those found against target cells loaded with peptide 290. Indeed, this fact can be explained due to the presence of additional and more active CTL epitopes on the virus. CTLs usually recognize peptide fragments from 8 to 11 amino acid residues in length bound to an MHC class I molecule (Falk et al., 1991 ), but it has also been described that longer peptides could be recognized by CTLs (Bertoletti et al., 1993
; Pauly et al., 1995
).
In conclusion, we have defined new T-cell epitopes on CSFV proteins. Peptide 290 on the NS23 protein has the capacity to elicit both CD4+ and CD8+ T-cell responses, suggesting that this protein segment may be a potential candidate for a synthetic peptide vaccine. Induction of CTL responses can be potentiated by covalent linkage of CTL and helper T-cell epitopes (Partidos et al., 1996 ; Shirai et al., 1994
; Stuhler & Walden, 1993
). Candidate sequences for synthetic peptide vaccines should be conserved among different CSFV strains, binding diverse MHC molecules (Kubo et al., 1994
; Oldstone et al., 1992
) and contain helper T-cell, CTL and B-cell epitopes (An & Whitton, 1997
).
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Acknowledgments |
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Received 22 August 2001;
accepted 23 November 2001.