Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas cedex, France
Correspondence
Abdenour Benmansour
abdenour{at}jouy.inra.fr
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ABSTRACT |
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Present address: Département de Biologie Cellulaire, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France.
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INTRODUCTION |
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Although in vitro assays for allo-antigen CTL responses have been described in catfish (Miller et al., 1986; Stuge et al., 2000
), carp (Fischer et al., 1998
) and rainbow trout (Fischer et al., 2003
), no assay for viral-antigen specific CTL is currently available for salmonid fish. In fact, the lack of specific tools still prevents in depth studies of the T-cell response to pathogens in teleosts. More importantly, monoclonal antibodies against fish TCR have never been obtained precluding phenotypic characterization and cell sorting. We therefore adapted a complementarity determining region (CDR) 3 length spectratyping methodology (immunoscope) to study the modifications of rainbow trout TCR
repertoire. This methodology provides an overview of the T-cell repertoire diversity in a given context, and has already been used to study mouse or human T-cell responses directed to a number of viruses including LCMV (Lin & Welsh, 1998
; Peacock et al., 2000
; Sourdive et al., 1998
), Theiler's virus (Kang et al., 2000
), C hepatitis virus (Umemura et al., 2000
), HIV (Kharbanda et al., 2000
; Kostense et al., 2001
; Kou et al., 2003
), B hepatitis virus (Sing et al., 2001
) and HTLV-1 (Saito et al., 2002
). In rainbow trout, CDR3 length spectratyping experiments revealed that VHSV induced both public and private specific T-cell responses in a rainbow trout clone (Boudinot et al., 2001
). In the present work, we used this methodology to identify the viral component responsible for the public T-cell response against the VHSV. We immunized fish with plasmid DNA expressing the VHSV G, and we analysed the modifications of the T-cell repertoire. We showed that G-based DNA vaccination induced a T-cell response reminiscent of the response to the virus, suggesting the public response observed during infection was directed to the G protein. The modifications of the T-cell repertoire due to DNA vaccination also revealed a substantial clonotypic diversity, which lasted for several months. Immunization with recombinant IHNV/VHSV chimeras generated through reverse genetics methodology further authenticated these results.
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METHODS |
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Leukocyte preparation.
Trout were sacrificed by over-exposure to 2-phenoxyethanol diluted 1 : 1000. The entire spleen was removed aseptically. Leukocytes were isolated by centrifugation through a Ficoll gradient (lymphocyte separation medium, d=1077; Eurobio), and used for RNA preparation.
DNA immunization.
Recombinant pcDNA_gVHS plasmid was constructed from the eukaryote expression vector pcDNA1 (Invitrogen). The G protein gene of VHSV (variant tr25; de Kinkelin et al., 1980) was reverse transcribed into cDNA from purified viral RNA. G-containing PCR product obtained with relevant primers was inserted under the control of the CMV promoter into pcDNA1. Plasmid DNA was prepared using the Endofree maxiprep kit (Qiagen), and resuspended in endotoxin-free water at 1 mg ml1 DNA concentration. Each fish was immunized by multipoint intramuscular injection with 50 µg plasmid, and boosted with the same amount on day 7 and 14. The challenge was performed on day 110, by VHSV intramuscular injection (07-71 strain, 50x106 p.f.u. per fish).
Recombinant IHNV/VHSV.
Recombinant IHNV deleted for the NonVirion (NV) gene and expressing the G/VSHV gene in place of G/IHNV was constructed from a full-length cDNA clone of IHNV (Biacchesi et al., 2000a). The pIHNV-
NV-eGFP-gVHSV plasmid was constructed from the previously described pIHNV-
NV-eGFP (Biacchesi et al., 2000a
). Briefly, SpeI and SmaI restriction enzyme sites were introduced by site directed mutagenesis at the start codon and at the end of the IHNV G gene, respectively. The VHSV G gene was recovered by RT-PCR from the VHSV RNA genome by using specific primers (Biacchesi et al., 2002
). Then, the IHNV G gene was deleted from pIHNV-
NV-eGFPSpeI/SmaI by SpeI and SmaI digestion and replaced with the VHSV G gene. The pIHNV-
NV-eGFP-gVHSV plasmid was used to recover recombinant chimera virus (rIHNV_gVHS) from transfected cells (Biacchesi et al., 2000b
). Similarly, the pIHNV-
NV-eGFP plasmid was used to produce a recombinant virus expressing the homologous G IHNV protein (rIHNV).
CDR3 length analysis.
The immunoscope methodology developed for mouse or human (Pannetier et al., 1995) was adapted for rainbow trout, using primers specific for trout V
1 to 4, J
and C
sequences. We chose the V
primers in-framework region (FR-2 region for V
1 and V
3 and FR-1 region for V
2 and V
4) to amplify most of the V
segments in each family. JB primers were designed to be specific for each of the J
1 to 9 segments except for J
4, because J
2 and J
4 sequences are almost similar. A J
2-4 primer was therefore designed in a region strictly similar in J
2 and J
4 to amplify both kinds of rearrangement with the same efficiency. Primer sequences are indicated in Table 1
. Immunoscope analysis was performed essentially as described in Boudinot et al. (2001)
. Briefly, PCR was performed on the relevant cDNA using V
- and C
-specific primers, which amplify sequences with a given V
, but with different CDR3 and J
s. In a second step, V
-C
PCR products were subjected to run-off reactions with different fluorescent C
- or J
-specific primers. Run-off products were loaded on to a polyacrylamide sequencing gel and size separated on an ABI 373 automated sequencer (Applied Biosystems). CDR3 length distributions were analysed using the immunoscope software, and the repertoire editing was performed using the ISEApeaks software (Collette & Six, 2002
).
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Statistical analysis.
The statistical significance of the presence of repeated junctions after vaccination or infection was assessed using Fisher's exact test. The significance of the frequency of a given CDR3 sequence in an immunized fish, compared to the control, was tested using a significance test of homogeneity of proportion with Yates's continuity correction, for specific repeated junctions.
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RESULTS |
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DISCUSSION |
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Interestingly, the G protein also constitutes the exclusive target of virus-neutralizing antibodies, which are sufficient to afford full protection against virus infection. Thus, protective B-cell and T-cell immune responses to VHSV appear to be focused on the viral G protein. Whether this observation is only coincidental or a sign of T-cell/B-cell cooperation remains difficult to address in rainbow trout. In fact, no CD4 homologue has been identified in this species, and there is no surface marker for helper T-cells. Although a CD8 homologue has been cloned and characterized (Hansen & Strassburger, 2000), there is still no validated specific antibody available for cell-sorting. T-cell responses identified using immunoscope methodology therefore integrate expansions of both helper and cytotoxic T lymphocytes. Notably, T-cell epitopes were identified in the glycoprotein of another rhabdovirus (rabies virus) in addition to neutralizing B-cell epitopes (Xiang et al., 1994
), but it was not demonstrated that T-cell response was formally required for protection.
The V4-J
1 response observed after three injections of pcDNA_gVHS was qualitatively different from the one observed after a prime/boost with the attenuated virus. The kinetics of the T-cell response to the DNA vaccine was much slower but it lasted for a longer time. Contrary to virus infection, no clear bias could be identified on day 21, although neutralizing antibodies were already detectable. Altered V
4-J
1 spectratypes were first detected on day 52, but the biases in profiles and junction distributions were less marked than after viral infection. The rainbow trout T-cell response induced by DNA immunization was therefore much slower than in the mouse (Palmowski et al., 2002
). It is well documented that the antibody response in trout is late compared with the mouse (Ellis, 1982
; Ingram, 1985
). In fact, it is not surprising that both arms of the specific immune response show a comparable time lag. In rainbow trout, the V
4-J
1 profiles were still strongly biased on day 110 after DNA immunization. In contrast, biased V
4-J
1 profiles returned to a bell-shaped form 3 weeks after a primary/unique infection with the attenuated virus (Boudinot et al., 2001
). This long-lasting bias following pcDNA_gVHS immunization was probably because of the persistence of G expression, which is well documented for DNA vaccination (Boudinot et al., 1998
; Heppell et al., 1998
). Accordingly, sustained expression of the antigen afforded by DNA vaccination did not initiate a clonal exhaustion of specific T cells (Ellis, 1982
; Ingram, 1985
; Palmowski et al., 2002
).
The V4-J
1 response elicited by pcDNA_gVHS or VHSV G-encoding viruses being most likely directed to the G protein, it provided a unique opportunity to examine the TCR
junction diversity against a protein in a teleost fish. The most frequent V
4-J
1 junction expanded after pcDNA_gVHS immunization or virus infection (Boudinot et al., 2001
) was the SSGDSYSE CDR3 and its variants. Notably, a non-V
, non-J
-encoded Asp residue is conserved in all variants, suggesting this residue is positively selected from randomly generated junctional sequences. Hence, this residue may be critical for TCRpeptide interaction. A significant redundancy, with a conserved consensus and variable positions, is therefore observed among junctions available for a given epitope in the rainbow trout naive repertoire. Such a variability of CDR3 sequences among TCR
rearrangements specific for a given epitope has been well documented for mouse T-cell responses against a single peptide (Bousso et al., 1998
; Lin & Welsh, 1998
). Two divergent 8 aa junctions SIGGLYSE and SPGQGNSE were also amplified in some pcDNA_gVHS-immunized fish, and could represent alternative responses to other G-protein epitopes. Although less prominent than the anti-G VHSV 8 aa V
4-J
1 response, 6 aa long V
4-J
1 junctions S(S/A)SYSE were expanded after pcDNA_gVHS immunization on day 52 or 110. All the amplified 6 aa junctions shared a SXS motif, and probably corresponded to TCRs specific for another epitope present on Novirhabdovirus G proteins.
When present, the SSGDSYSE junction was always amplified more than other G-specific 8 aa CDR3 junctions, suggesting that SSGDSYSE would lead to the highest TCR avidity among S(S/N)(G/R/Q)DSYSE junctions. This is reminiscent of the consistent and overall reproducible hierarchy of T-cell responses observed in the mouse (Chen et al., 2000; Yewdell & Bennink, 1999
). However, we also observed that TCR usage following pcDNA_gVHS immunization differed from animal-to-animal, even if they shared the same genetic background, a feature also described in the mouse (Blattman et al., 2000
; Bousso et al., 1998
; Lin & Welsh, 1998
). Thus, TCR
repertoire is diverse enough in rainbow trout, so that the specific TCRs of the dominant T-cell clones may be unique to the individual. This appears to be a common feature among vertebrates, which could partly explain the heterogeneous sensitivity of genetically identical hosts to the same virus.
Several anti-VHS glycoprotein V4-J
1 junctions were observed in pcDNA_gVHS-immunized fish, suggesting that the response was more diversified than after virus infection. This diversity was also maintained after VHSV-challenge of pcDNA_gVHS-immunized fish (data not shown). We retrieved two or three different clonal V
4-J
1 expansions in each fish, suggesting that the V
4-J
1 memory response maintained its initial diversity and was probably directed against several epitopes. By contrast, after prime/boost with attenuated VHSV, we detected expansion of only one unique V
4-J
1 junction per fish. This may indicate that the infectious context narrows the memory response during the first virus encounter, while the DNA vaccination would induce weaker competition between specific T cells and would keep higher diversity. A higher clonotypic diversity in the response to the genetic vaccine may also be promoted by inflammatory signals present in the plasmid sequence. However, the plasmid backbone used (pcDNA1) does not contain immuno-stimulatory motifs. Such differences in T-cell epitopes recognized after immunization versus after infection have been observed in mammals, and ascribed to different contexts of T-cell competition and peptide presentation (Palmowski et al., 2002
; Vogel et al., 2002
). Finally, this hypothesis would be consistent with the great capacity of DNA vaccines to induce highly efficient cellular responses, and protection against pathogens. Indeed, the diversity of available specific T-cell clones has a critical influence on the protective capacity of the cellular response to pathogens (Messaoudi et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 26 March 2004;
accepted 19 May 2004.