The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout

Pierre Boudinot, David Bernard, Samira Boubekeur{dagger}, Maria-Isabel Thoulouze, Michel Bremont and Abdenour Benmansour

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
T-cell responses to viruses are still poorly investigated in lower vertebrates. In rainbow trout, a specific clonal expansion of T cells in response to infection with viral haemorrhagic septicaemia virus (VHSV) was recently identified. Expanded T-cell clones expressed a unique 8 aa V{beta}4-J{beta}1 junction (SSGDSYSE) in different individuals, reminiscent of a typical public response. To get further insight into the nature of this response the modifications of the T-cell repertoire following immunization with plasmid expressing the VHSV external glycoprotein (G), which is the only protein involved in protective immunity, was analysed. After G-based DNA immunization, CDR3-length spectratypes were skewed for several V{beta}-J{beta} combinations, including V{beta}4-J{beta}1. In V{beta}4-J{beta}1, biases consisted of 6 and 8 aa junctions that were detected from day 52, and were still present 3 months after DNA immunization. Sequence analysis of the V{beta}4-J{beta}1 junctions showed that the 8 aa junction (SSGDSYSE) was clearly expanded, indicating that viral G protein was probably the target of the anti-VHSV public response. Additional 6 and 8 aa V{beta}4-J{beta}1 junctions were also expanded in G-DNA-vaccinated fish, showing that significant clonotypic diversity was selected in response to the plasmid-delivered G protein. This higher clonotypic diversity may be related to the demonstrated higher efficiency of G-based DNA vaccines over whole virus immunization. The use of infectious hematopietic necrosis virus (IHNV) recombinant viruses, expressing the VHSV G protein, further substantiated the VHSV G-protein specificity of the 8 aa V{beta}4-J{beta}1 response and designated the 6 aa V{beta}4-J{beta}1 response as potentially directed to a T-cell epitope common to VHSV and IHNV.

{dagger}Present address: Département de Biologie Cellulaire, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are fish rhabdoviruses classified in the genus Novirhabdovirus, responsible for systemic diseases costly for the salmonid farming industry. Six proteins are encoded by their non-segmented RNA genomes (Basurco & Benmansour, 1995; Benmansour et al., 1994; Bernard et al., 1990; Kurath et al., 1985; Kurath & Leong, 1985; Schütze et al., 1995, 1999; Thiry et al., 1990). Like in other rhabdoviruses, the glycoprotein (G) is the only protein exposed at the surface of VHSV and IHNV, and is responsible for the entry of the virus into the cell by receptor-mediated endocytosis. The G protein is the target of neutralizing antibodies, which constitute the main component of the fish protective response against rhabdoviruses (Engelking & Leong, 1989; Lorenzen et al., 1990). Although VHSV and IHNV are structurally similar and evolutionary related, their G protein sequences have only 50 % aa identity, and typically share only short common peptides 10–15 aa long in distinct regions of the protein. Moreover, neither VHSV nor IHNV can induce cross-protection against each other. The gene encoding the G protein of the two viruses has been cloned into several vectors to produce recombinant proteins in different cell systems. However, vaccination with the recombinant G proteins did not protect as well as when using the intact virus, probably because of inappropriate folding. A role for other viral proteins in the protection was also evoked. This issue was finally settled when several teams demonstrated that a high level of protection was consistently observed after immunization with plasmids encoding the G protein (Anderson et al., 1996; Boudinot et al., 1998; Heppell et al., 1998). By contrast, vaccination with plasmids encoding other viral proteins was not successful, which confirmed that anti-G neutralizing antibodies were mandatory to protect fish against a viral challenge (Corbeil et al., 1999). DNA vaccination was shown to induce both specific and non-specific immune responses (Boudinot et al., 1998; Kim et al., 2000). The G-specific antibody response and the non-specific mechanisms induced by genetic immunization in rainbow trout have been well studied (Lorenzen et al., 2002). In contrast, the putative role of an anti-G T-cell response in establishment of protection has never been documented.

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{beta} 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fish.
Rainbow trout were raised in the fish facilities of Institut National de la Recherche Agronomique (Jouy-en-Josas). Homozygous trouts were obtained by gynogenesis from the so-called ‘INRA synthetic strain’ population as described in Diter et al. (1993). Some of the gynogenetic animals were subjected to a methyl-testosterone treatment and developed as homozygous neomales. The rainbow trout heterozygous ‘clone’ EQ2 was obtained by crossing such a neomale with a homozygous female from a different gynogenesis experiment. Animals within this ‘clone’ were therefore heterozygous, but shared the same genetic background.

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 ml–1 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-{Delta}NV-eGFP-gVHSV plasmid was constructed from the previously described pIHNV-{Delta}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-{Delta}NV-eGFPSpeI/SmaI by SpeI and SmaI digestion and replaced with the VHSV G gene. The pIHNV-{Delta}NV-eGFP-gVHSV plasmid was used to recover recombinant chimera virus (rIHNV_gVHS) from transfected cells (Biacchesi et al., 2000b). Similarly, the pIHNV-{Delta}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{beta}1 to 4, J{beta} and C{beta} sequences. We chose the V{beta} primers in-framework region (FR-2 region for V{beta}1 and V{beta}3 and FR-1 region for V{beta}2 and V{beta}4) to amplify most of the V{beta} segments in each family. JB primers were designed to be specific for each of the J{beta}1 to 9 segments except for J{beta}4, because J{beta}2 and J{beta}4 sequences are almost similar. A J{beta}2-4 primer was therefore designed in a region strictly similar in J{beta}2 and J{beta}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{beta}- and C{beta}-specific primers, which amplify sequences with a given V{beta}, but with different CDR3 and J{beta}s. In a second step, V{beta}-C{beta} PCR products were subjected to run-off reactions with different fluorescent C{beta}- or J{beta}-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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Table 1. Primers for rainbow trout immunoscope analysis

 
TCR{beta} junctions.
To study the sequence composition of expanded peaks identified in immunoscope profiles, we performed PCR amplification from the relevant cDNA, using the corresponding VB and CB primers. PCR products were purified using Sephacryl S-400 columns (Pharmacia Biotech) and cloned into the TOPO-TA cloning system (Invitrogen). Several colonies selected at random were grown overnight in LB plus ampicillin broth, and the plasmid purified with a plasmid miniprep spin kit (Nucleospin; Macherey-Nagel). Purified plasmids were subjected to automated sequencing with direct and reverse universal primers. The genetic computer group package (GCG; Madison) was used for sequence alignment.

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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
G-VHSV DNA vaccination induced modifications of the T-cell repertoire
Effective protection against VHS could be obtained through vaccination with live viral vaccines or through immunization with a plasmid DNA expressing the virus G protein. The protection was always correlated with the presence of specific-neutralizing antibodies directed to the G protein. We have recently shown that VHSV infection also induced a specific T-cell response shown through an important and specific modification of the T-cell repertoire (Boudinot et al., 2001). To investigate if this T-cell response was directed to the G protein we used plasmid DNA vaccination. The plasmid pcDNA_gVHS (Boudinot et al., 1998), which contains the entire gene of the G protein, or the empty plasmid pcDNA1 were used to immunize two groups of rainbow trout. To avoid the effect of fish-to-fish genetic variability, and to facilitate comparative analysis of the response induced by the virus and by the DNA vaccine, we used the same clone of rainbow trout, as in our previous study (clone EQ2). Each rainbow trout was injected on day 1, 7 and 14 with 50 µg of the plasmids. In vivo G-protein expression was confirmed by the detection of specific-neutralizing antibodies. For this purpose, sera prepared from fish sacrificed for T-cell repertoire analysis were subjected to a plaque neutralization assay. We observed high titres of neutralizing antibodies on day 52 in fish immunized with pcDNA_gVHS, but not in fish injected with the empty plasmid, showing that the antigen was successfully processed and recognized by the fish immune system (data not shown). To investigate the T-cell response induced by DNA vaccination, we analysed the repertoire of T cells from vaccinated and control fish. We used a spectratyping method adapted to display the CDR3 length profiles of the V{beta} chain of the rainbow trout TCR (Boudinot et al., 2001). Two fish immunized with pcDNA_gVHS and two fish immunized with pcDNA1 were sacrificed at day 7, 21 and 52 post-immunization. CDR3 length profile analysis was performed for each time point. Complementary DNA was synthesized from spleen RNA, and PCR-amplified using VB1 to 4 as forward primers and a C{beta}-specific oligonucleotide (CB2) as the reverse primer. Each VB-CB PCR product was then subjected to a run-off reaction with internal JB1 to 9 fluorescent primers specific to J{beta}1 to 9 segments. On day 7 and 21, only bell-shaped profiles typical of a naive repertoire were observed in fish immunized with either pcDNA_gVHS or pcDNA1 (data not shown). On day 52 post-immunization, we observed significant modifications of profiles for several V{beta}-J{beta} combinations in pcDNA_gVHSV-immunized fish but not in the plasmid control group. Biased combinations included V{beta}3-J{beta}3, V{beta}4-J{beta}1, V{beta}4-J{beta}3, V{beta}4-J{beta}5, V{beta}4-J{beta}7 and V{beta}4-J{beta}8. The response to the G-DNA vaccine seemed to be specially skewed towards V{beta}4-expressing TCR{beta} rearrangements, including the V{beta}4-J{beta}1 combination involved in the public response to the virus, i.e. the response observed in all individuals sharing the same genetic background. Typical V{beta}1-4/J{beta}1-9 immunoscope profiles obtained on day 52 from a pcDNA_gVHS-immunized individual are shown in Fig. 1. These results strongly suggest that DNA immunization with a VHSV G-protein expression plasmid induced a specific T-cell response in rainbow trout.



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Fig. 1. Immunoscope profiles of pcDNA_gVHS-immunized rainbow trout (day 52). cDNA from the spleen leukocytes was amplified using VB1 to 4 and CB2 primer, and PCR products subjected to run-off reactions using fluorescent JB1 to JB9 primers. After separation of the run-off reactions in a sequencing gel, the fluorescent profiles were analysed with immunoscope and ISEApeaks software. Each profile represents the CDR3 length diversity of a given V{beta}-J{beta} combination. Fragment length is on the x-axis, and fluorescence intensity is on the y-axis.

 
pcDNA_gVHS immunization induced the public response observed after VHSV infection
G-protein expression obtained through DNA immunization induced a significant bias in the V{beta}4-J{beta}1 combination. This combination was previously shown to participate to a specific public response during VHSV infection in the EQ2 rainbow trout clone. To get further insight into the T-cell response induced by G expression, we compared the modifications of the V{beta}4-J{beta}1 profiles following DNA immunization with pcDNA_gVHS or with pcDNA1 at various time points. V{beta}4-J{beta}1 profiles were skewed in pcDNA_gVHS-immunized fish on day 52, while they remained unchanged in control fish (Fig. 2). Depending on the individuals, either one peak corresponding to a CDR3 of 8 aa (trout 4) or two peaks corresponding to CDR3 of 6 and 8 aa (trout 5) were significantly amplified.



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Fig. 2. V{beta}4-J{beta}1 CDR3 length profiles after DNA vaccination. Fishes were injected with 50 µg pcDNA_gVHS or control pcDNA1 on day 1, 7 and 14. Immunoscope analysis was performed on day 21 (a) and on day 52 (b). Spleen cDNA from relevant fish was amplified with VB4 and CB2 primers, and PCR products subjected to a run-off reaction using fluorescent JB1 primer and analysed as in Fig. 1.

 
To analyse further V{beta}4-J{beta}1 junctions corresponding to biased immunoscope profiles we amplified and cloned V{beta}4-J{beta}1 rearrangements from spleen cDNA of trout immunized with pcDNA_gVHS. For each individual, several clones were picked at random and sequenced. Different V{beta}4-J{beta}1 junctions were present more than once in pcDNA_gVHS-immunized trout, suggesting that DNA-vaccine induced V{beta}4-J{beta}1 T-cell response (Fig. 3). The SSGDSYSE junction (8 aa long CDR3) was present in 7 of 11 clones with the same CDR3 length as in the pcDNA_gVHS-immunized trout 4. This junction was also highly amplified in response to a non-lethal VHSV infection (Boudinot et al., 2001). This finding strongly suggests that the viral G protein was also responsible for the V{beta}4-J{beta}1 public response observed following VHSV infection. In trout 5, we observed two different amplified junctions with an 8 aa long CDR3, SNRDSYSE in 2 of 11 clones and SPGQGNSE in 3 of 11 clones. The first one was clearly reminiscent of the highly amplified SSGDSYSE junction. Two different junctions with a CDR3 size of 6 aa were also found more than once in trout 5, SASYSE in 3 of 7 clones and SSSYSE in 2 of 7 clones. These two 6 aa long junctions were encoded by the same nucleotide sequence, suggesting that their repetition may result from clonal T-cell expansion, and not from codon redundancy for serine residue. This observation was consistent with the corresponding V{beta}4-J{beta}1 profile, which had two amplified peaks corresponding to 6 and 8 aa long CDR3 (Fig. 2b, trout 5). Interestingly, the junction SSSYSE was also present once among sequences from the pcDNA_gVHS-immunized trout 4 (Fig. 3). V{beta}4-J{beta}1 rearrangements from spleen cDNA of trout immunized with pcDNA1 were also sequenced. Among 44 and 30 V{beta}4-J{beta}1 junctions from two control fish all were present only once and all were different from junctions amplified in pcDNA_gVHS-immunized fish (data presented for one control fish; Fig. 3, trout 6). Taken together, these observations suggest that DNA immunization with a plasmid encoding the VHSV G protein triggers the V{beta}4-J{beta}1 public response identified after viral infection. The 8 aa junction SSGDSYSE was expanded most frequently, but a variant junction (SNRDSYSE) may be used instead. Also, TCR{beta} with 6 aa CDR3 was selected by the G-DNA vaccination.



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Fig. 3. CDR3 sequences from the profiles shown in Fig. 2(b). Spleen cDNA from pcDNA_gVHS- or pcDNA1-immunized fish was amplified using VB4 and JB1 primers (pcDNA_gVHS: trout 4 and 5, pcDNA1: trout 6). PCR products were purified and cloned, and clones selected at random were sequenced. Nucleotide and amino acid sequences of the in-frame junctions were sorted according to their CDR3 length. Underlined nucleotides correspond to the D{beta} sequence. Boldfaced nucleotides are non-coded in the germline, and most probably represent N additions. Nucleotides in italic are compatible with P addition mechanism. Nucleotides in bold and italic represent either P or N additions. Out-of-frame junctions were not represented in the figure (5 sequences for trout 4, 6 sequences for trout 5 and 9 sequences for trout 6). The proportion of unique versus repeated junctions was significantly different in trout 4 and 5 from that in control trout 6 (P<0·01 and 0·001 in a Fisher's exact test, respectively). Asterisk (*), indicates that the frequency of a junction in an immunized fish is significantly different from that in the control trout 6 in a significance test of homogeneity of proportion with Yates's continuity correction (confidence level 5 %).

 
pcDNA_gVHS immunization induced a long-lasting T-cell response
DNA vaccination is known to ensure sustained expression of antigens (Donnelly et al., 1997; Heppell et al., 1998). For G-VHSV, we have previously shown that pcDNA_gVHS was still detectable in muscle tissue 7 weeks post-injection (Boudinot et al., 1998), and G-protein expression was detected 3 weeks after immunization (Lorenzen et al., 1998). To investigate the effects of the sustained expression of the G protein, we performed a CDR3 spectratyping analysis 3 months after immunization. V{beta}4-J{beta}1 profiles from fish sacrificed on day 110 after pcDNA_gVHS immunization were still clearly biased with expanded peaks for 6 and 8 aa CDR3 (Fig. 4, trout 7 and 8). The CDR3 length profile of V{beta}4-J{beta}1 transcripts was also analysed in two control fish on day 110 after immunization with the empty plasmid, and showed a typical unbiased bell-shaped distribution (data presented for one control fish; Fig. 4, trout 9). To verify that the expanded peaks were due to an amplification of G-specific V{beta}4-J{beta}1 junctions, we cloned the V{beta}4-J{beta}1 rearrangements from the corresponding spleen cDNAs. Thirty and 31 junctions were analysed from trout 7 and 8, respectively (Fig. 5). The 8 aa junction (SSGDSYSE) was highly amplified in trout 7 (8 of 12 junctions of the same length). A closely related sequence (SSRDSYSE) was observed once. The 6 aa junction SSSYSE was also clearly amplified in the same fish (9 of 10 junctions of 6 aa), confirming results from day 52. Thus, the biased profile observed in trout 7 was due to the high frequency of 6 and 8 aa junctions already expanded after viral infection or after DNA immunization on day 52. The bias was less pronounced but still clear in trout 8, where two 8 aa CDR3s were amplified: SSQDSYSE, similar to the SSGDSYSE found in trout 7, and SIGGLYSE, which had not been found previously. Taken together, these results showed that DNA immunization with a plasmid encoding the VHSV G maintains a long-lasting modification of the V{beta}4-J{beta}1 transcript repertoire, since biases lasted for at least 3 months. The 8 aa SSGDSYSE junction and its variants were systematically and strongly amplified in response to the VHSV G protein, while amplification of the 6 aa long SSSYSE junction was genuine but less regular. Finally, the distribution of expanded junctions was variable from fish-to-fish, although they all shared the same genetic background.



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Fig. 4. V{beta}4-J{beta}1 CDR3 length profiles 3 months after DNA immunization. Fishes were injected with 50 µg pcDNA_gVHS (trout 7 and 8) or control pcDNA1 (trout 9) on day 1, 7 and 14, and immunoscope analysis was performed on day 110. Spleen cDNA from relevant fish was amplified with VB4 and CB2 primers, and PCR products subjected to a run-off reaction using fluorescent JB1 primer. Immunoscope profiles were obtained and edited as described for Fig. 1.

 


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Fig. 5. CDR3 sequences from the skewed profiles identified in Fig. 4 (trout 7 and 8). Spleen cDNAs from day 110 trout 7 and 8 were amplified using VB4 and JB1 primers. PCR products were purified and cloned, and clones selected at random were sequenced. Nucleotide and amino acid sequences of the in-frame junctions were sorted according to their CDR3 length and edited as in Fig. 3. Out-of-frame junctions were not represented (5 sequences for both trout 7 and 8). The proportion of unique versus repeated junctions was significantly different in trout 7 and 8 from that in control trout 6 (P<0·001 and 0·01 in a Fisher's exact test, respectively). Asterisk (*), indicates that the frequency of a junction in an immunized fish is significantly different from that in the control trout 6 in a significance test of homogeneity of proportion with Yates's continuity correction (confidence level 5 %).

 
Recombinant IHNV expressing VHSV G protein induced the same V{beta}4-J{beta}1 T-cell response
To confirm further the G specificity of the V{beta}4-J{beta}1, we used a chimeric recombinant IHNV, where the IHNV G gene was replaced by the gene encoding the VHSV G protein (rIHNV_gVHS). A recombinant virus expressing the homologous IHNV G protein was also used in parallel (rIHNV). In both viruses, the green fluorescent protein gene replaced the non-structural NV gene. This replacement resulted in a strong reduction of virulence (data not shown), allowing non-lethal infection of juvenile fish. Fishes were injected with 5x105 p.f.u. of rIHNV_gVHS or rIHNV. On day 27, they were subjected to a second injection with the respective virus, and the V{beta}4-J{beta}1 was analysed on day 50. Immunoscope analysis was performed on two rIHNV_gVHS-infected animals. One showed a highly biased profile at an 8 aa CDR3 size (trout 10), the second displayed a non-biased profile, although anti-VHSV neutralizing antibodies were detected in both fish (data not shown). The unexpected defect of the T-cell response to rIHNV_gVHS in one trout was probably due to the important biological differences of the recombinant virus compared with wild-type VHSV. Interestingly, when run-off reactions were performed using an internal C{beta}-specific primer on different VB-CB1 PCR products, several biases were observed (data not shown). These biases may correspond to private responses against VHSV G, but most probably represented responses against IHNV proteins. Skewed spectratypes were also observed in two rIHNV-infected fish, which were biased for several CDR3 sizes of 6 and 8 aa for one fish and 7 aa for the second fish. Fig. 6 exemplifies biased V{beta}4-J{beta}1 profiles from trout infected with rIHNV_gVHS (expanded peak for 8 aa CDR3) or rIHNV (expanded peaks for 6 and 8 aa CDR3). V{beta}4-J{beta}1 rearrangements corresponding to the profiles shown in Fig. 6 were amplified from the respective spleen cDNA, and the junctions were sequenced. Junction SSGDSYSE was found 6 of 8 times with 8 aa CDR3 in trout 10 (Fig. 7), confirming that infection with rIHNV_gVHS induced expansion of the same junction as identified after G-VHSV DNA vaccination or VHSV infection. Sequence analysis was also performed on trout 11, infected with rIHNV. Consistent with the profile shown in Fig. 6, both 6 and 8 aa CDR3 junctions were observed several times. The 6 aa amplified junction SSSYSE, which was expanded after G-VHSV DNA immunization, was observed twice (Fig. 7). Thus, this junction might correspond to a TCR specific for an epitope common to VHSV and IHNV G proteins. VHSV and IHNV G proteins have several sequence similarities and complete sequence identity in a few short regions. In trout 11, the amplified 8 aa junction SGGISYSE was present 3 of 4 times. It was never observed after VHSV immunization and most probably should correspond to an IHNV epitope. These results illustrate that a recombinant virus expressing VHSV G is able to induce the same V{beta}4-J{beta}1 public T-cell response as VHSV G DNA immunization or VHSV infection.



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Fig. 6. V{beta}4-J{beta}1 CDR3 length profiles 3 months after infection with rIHNV_gVHS or with rIHNV. Fishes were infected on day 1 and 27. Immunoscope analysis was performed on cDNA from spleen leukocytes on day 42. Spleen cDNA from trout 10 (rIHNV_gVHS) and 11 (rIHNV) was amplified with VB4 and CB2 primers, and PCR products subjected to a run-off reaction using fluorescent JB1 primer. Immunoscope profiles were obtained and edited as described for Fig. 1.

 


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Fig. 7. CDR3 sequences from the V{beta}4-J{beta}1 profiles shown in Fig. 6. V{beta}4-J{beta}1 junctions corresponding to trout 10 and 11 of Fig. 6 were amplified, cloned and several clones sequenced. Nucleotide and amino acid sequences of the in-frame junctions were sorted according to their CDR3 length and edited as in Fig. 3. Out-of-frame junctions were not represented in the figure (4 sequences from trout 10 and 1 sequence from trout 11). The proportion of unique versus repeated junctions was significantly different in trout 10 and 11 from that in control trout 6 (P<0·01 in a Fisher's exact test, respectively). Asterisk (*), indicates that the frequency of a junction in an immunized fish is significantly different from that in the control trout 6 in a significance test of homogeneity of proportion with Yates's continuity correction (confidence level 5 %).

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have recently shown that VHSV infection induced a specific T-cell response shown through important and specific modifications of the T-cell repertoire (Boudinot et al., 2001) with a public V{beta}4-J{beta}1 component. In this work, we showed that a V{beta}4-J{beta}1 T-cell response was induced by G-based DNA vaccine or by chimeric virus expressing G-VHSV. These results supported the idea that the V{beta}4 public response elicited by VHSV infection was directed to the viral G protein. The V{beta}4-J{beta}1 profile was altered on day 52 or 110 after DNA vaccination, with expanded peaks corresponding to CDR3 sizes of 8 and 6 aa. Sequencing analysis confirmed that the biases observed after virus infection and DNA vaccination corresponded to the expansion of similar junctions in fish sharing the same genetic background. Thus, the SSGDSYSE junction was clearly expanded after DNA vaccination, as it was after a prime/boost with an attenuated VHSV (Boudinot et al., 2001). Although not direct, this is a strong indication that the VHSV G protein contains the target of the dominant public V{beta}4-J{beta}1 response to the virus. The identification of VHSV glycoprotein as the target of the V{beta}4-J{beta}1 public response was further confirmed by the analysis of the T-cell response to recombinant IHNV expressing the VHSV glycoprotein in place of the homologous glycoprotein. Indeed, the 8 aa junction SSGDSYSE was found clearly amplified in a single trout subjected to a prime/boost protocol with rIHNV_gVHS. As expected, rIHNV also induced bias of the T-cell repertoire. The response consisted of important bias of the profiles for CDR3 size of 6, 7 or 8 aa, which was further confirmed by sequence determination of the junctions. Most importantly, not a single 8 aa long CDR3 characteristic of the VHSV dominant response was retrieved. The modifications of the V{beta}4-J{beta}1 repertoire induced by the two glycoproteins expressed on the same viral backbone were therefore clearly different regarding the dominant response. However, the 6 aa junction SSSYSE was amplified after rIHNV infection, as well as after immunization with VHSV or VHSV glycoprotein. Therefore, this junction may correspond to a TCR specific for an epitope common to VHSV and IHNV glycoproteins. VHSV and IHNV glycoproteins have several sequence similarities and complete sequence identity in a few short regions. Such T-cell cross-reactivity has been observed for other viruses (Oldstone et al., 2001), and can accommodate the selection of common memory T cells by several successive viral infections (Selin et al., 1999).

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 V{beta}4-J{beta}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{beta}4-J{beta}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{beta}4-J{beta}1 profiles were still strongly biased on day 110 after DNA immunization. In contrast, biased V{beta}4-J{beta}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 V{beta}4-J{beta}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{beta} junction diversity against a protein in a teleost fish. The most frequent V{beta}4-J{beta}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{beta}, non-J{beta}-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 TCR–peptide 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{beta} 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{beta}4-J{beta}1 response, 6 aa long V{beta}4-J{beta}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{beta} 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 V{beta}4-J{beta}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{beta}4-J{beta}1 expansions in each fish, suggesting that the V{beta}4-J{beta}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{beta}4-J{beta}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).


   ACKNOWLEDGEMENTS
 
We thank A. Colette, A. Six and P.-A. Cazenave for expert advice and for providing the ISEApeaks software. J. Kanellopoulos for helpful discussions. B. Buteau, F. Coulpier and the staff of the experimental fish facilities are also acknowledged for their excellent technical assistance. Fish clone EQ2 was provided by M. Dorson and E. Quillet. This work was supported by the Institut National de la Recherche Agronomique and European Community Project FAIR 98-4026.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 26 March 2004; accepted 19 May 2004.