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Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
Correspondence
Barbara A. Blacklaws
bab2{at}cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: AFSSA Maisons-Alfort, LERPAZ, UMR 1161 Virology, 23 avenue du General de Gaulle, 94706 Maisons-Alfort cedex, France. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). VISNA is a member of the subfamily Lentivirinae, which includes human, simian, feline and bovine immunodeficiency viruses (HIV, SIV, FIV and BIV, respectively), equine infectious anaemia virus (EIAV) and the closely related caprine arthritis encephalitis virus (CAEV). Unlike HIV and other immunodeficiency viruses, VISNA infects cells of the monocyte/macrophage lineage and dendritic cells but not T lymphocytes (Gendelman et al., 1985, 1986; Gorrell et al., 1992; Ryan et al., 2000). Due to the tropism for mononuclear phagocytes, lymphoid tissues are considered to be important targets for the replication of VISNA (Blacklaws et al., 1995a; Pétursson et al., 1991). This restriction in tropism makes VISNA a unique model for understanding immunity to and pathogenesis of lentiviruses in the absence of T-cell infection in the infected host.
Cytotoxic T-lymphocytes (CTL) play an important role in immune control of both acute and chronic viral infections and are key immunological mediators of viral clearance (Harty et al., 2000; Wong & Pamer, 2003). In lentiviral infections, control of replication of HIV and SIV has been linked strongly to robust specific CD8+ CTL although CD4+ T-lymphocyte responses and antibodies are also important (Benito et al., 2004; Heeney & Plotkin, 2006; Maecker & Maino, 2003; McMichael & Rowland-Jones, 2001); protection in an FIV vaccination model has been linked to T-lymphocyte responses (Flynn et al., 1996; Hosie et al., 1998; Pu et al., 1997); and CTL are also associated with control of virus replication in horses infected with EIAV (McGuire et al., 2004). It would therefore be reasonable to assume that protective mechanisms in VISNA will involve CTL.
Although we have shown the presence of precursor CTL and ex vivo directly active CTL in VISNA-infected sheep previously (Bird et al., 1993; Blacklaws et al., 1994, 1995b) the proteins of the virus recognized have not been determined. In this study, we have shown that all VISNA proteins except for TAT are recognized by CTL and that POL contained a cluster of epitopes in its central region (aa 612–636). We have also shown that a DNA-prime–Modified vaccinia virus Ankara (MVA)-boost vaccination regime is successful in inducing a CTL response in sheep. Given the restricted tropism of VISNA, this new information on the epitope specificity of CD8+ CTL against VISNA is relevant to our understanding of CD8+ T-cell recognition of infected macrophages and dendritic cells in all lentiviral infections.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) for >3 months before use. Finnish LandracexDorset crosses (B1125 and B1139 only) were given 105 TCID50 virus subcutaneously; Friesland and Lleyn sheep were infected intratracheally with 105 and 103 TCID50 VISNA, respectively. Sheep used for preparation of heterologous skin cells used in the CTL assays were BF1 (Black Face), C111 and D142 (Lleyn). Chromium release assays were used to show that heterologous skin cells did not present VISNA antigen to CTL of sheep under study (data not shown). All sheep were kept in the animal facilities of the Department of Veterinary Medicine, University of Cambridge and were used in accordance with procedures laid out in the Animals (Scientific Procedures) Act (1986) of the UK.
Viruses and antibodies.
VISNA strain EV1 is the British isolate of VISNA (Sargan et al., 1991) and viral stocks were grown and titred in sheep skin cells (Bird et al., 1993).
MVA was obtained from Professor G. Smith, Imperial College, London, UK. Recombinant MVA (rMVA) viruses expressing all VISNA genes were constructed within our laboratory using the transfer plasmid pSC11 (Chakrabarti et al., 1985). The intermediate transfer plasmid pSC11env was a kind gift from Dr D. Rodríguez, Centro Nacional de Biotecnologia-CSIC, Madrid, Spain (Sánchez et al., 2002). Other plasmids were constructed using PCR products from Hirt prepared DNA (gag, pol, env, vif, gag p17, gag p25 and gag p14) or from cDNA (tat and rev) of VISNA EV1-infected cells, using primers to add start and stop codons as necessary. Six overlapping fragments of the pol gene were amplified by PCR with the pol gene clone [pBluescriptSKpol1.13, pol insert sequence (GenBank accession no. EU528031
[GenBank]
) used in full-length pol rMVA] as template. Primers were as follows for individual fragments (nucleotide positions in pol are indicated in parentheses; F, forward primer; R, reverse primer; BamHI sites are underlined, start codons in bold): fragment 1 (1–608), PolF4 5'-AAGGATCCACCATGCATACAGCTGCAGGGAAA-3', PolR4 5'-TGGATCCAAGCTTCAATTCCCTGAAGTCTATTAACAT-3'; fragment 2 (538–1126), PolF5 5'-AAGGATCCACCATGAGCACTCCTATATTTTGCATC-3', PolR5 5'-TGGATCCAAGCTTTGGTCCTTCTTCCATATCTG-3'; fragment 3 (1063–1677), PolF6 5'-AAGGATCCACCATGGAACTACATCCAGAGAGATG-3', PolR6 5'-TGGATCCAAGCTTGTATAATATGTTGGCCCCTC-3'; fragment 4 (1609–2176), PolF7 5'-AAGGATCCACCATGAGGGGATCAGTAAGATGGAA-3', PolR7 5'-TGGATCCAAGCTTCCCTATCATAGCCCATTGATT-3'; fragment 5 (2104–2793), PolF8 5'-AAGGATCCACCATGGCAGGACAAGTAAAGAAGAT-3', PolR8 5'-TGGATCCAAGCTTTTCTGCTATAAATGCTGGTCC-3'; fragment 6 (2719–3267), PolF9 5'-AAGGATCCACCATGACAACTATGAAGTGGTATGC-3', PolR9 5'-TGGATCCAAGCTTAGGCCCTATCTCCCTATTT-3'. PCR products were cloned into a cloning vector for sequencing, then the relevant insert was subcloned into the SmaI site of pSC11 and the correct orientation was confirmed by restriction enzyme digestion and sequencing. Recombinant viruses were made as described previously (Sánchez et al., 2002), routinely grown in QT35 cells (fibrosarcoma cells from Japanese quail, ECACC reference no. 93120832) and the titre determined by plaque assay (p.f.u.) using β-galactosidase staining for recombinant virus plaques. Expression of VISNA genes was confirmed by Western blotting and/or RT-PCR (data not shown). rMVA expressing full-length gag was called MVAgag, full-length pol MVApol, env MVAenv, tat MVAtat, rev MVArev, vif MVAvif, and gag subfragments: matrix p17 MVAp17, capsid p25 MVAp25 and nucleoprotein p14 MVAp14. The rMVAs expressing fragments of pol were called MVApolfragment1–6. A rMVA was also generated using pSC11 alone (MVApSC11) to act as a control virus expressing β-galactosidase.
Monoclonal antibody (mAb) VPM19 (Hopkins & Dutia, 1990), specific for ovine major histocompatibility complex (MHC) class I antigens (mouse IgG1), was grown and purified in our laboratory, and purified mAb IL-A88 (Toye et al., 1990), specific for bovine MHC class I antigens (mouse IgG2a), was kindly provided by Dr S. Ellis (Institute for Animal Health, Compton, UK).
Generation of DNA vaccines.
Using pBluescriptSKpol1.13 plasmid DNA as template, full-length pol and pol gene fragment 4 (positions 1609–2175 in pol) were amplified by PCR using the Expand Long Template PCR System kit or Pwo DNA polymerase (both by Roche), respectively. The amplified full-length VISNA pol gene and pol fragment 4 were inserted into the BamHI site of pcDNA3.1his/V5 (Invitrogen) and called pcDNA3.1pol
and pcDNA3.1polfrag4, respectively. To generate a full-length POL fusion protein expressing the V5 tag for testing expression of the protein, the stop codon in the full-length pol gene was deleted by XbaI digestion using an internal pol gene XbaI site and the pcDNA3.1/His-V5 multiple cloning XbaI site, causing the deletion of a 78 bp 3' fragment of the pol gene including the stop codon. The correct construction of pcDNA3.1pol and pcDNA3.1polfrag4 was confirmed by sequencing; then plasmid DNA was used to transfect nearly confluent QT35 monolayers using Fugene6 (Roche) in Glasgow's modified Eagle's medium (GMEM) supplemented with 2 % fetal calf serum (FCS), 10 % tryptose phosphate broth (TPB: 2 % bacto tryptose, 0.2 % bacto dextrose, 0.5 % NaCl, 0.25 % Na2PO4), 100 U penicillin and 100 µg streptomycin ml–1 (2 %FCS/TPB/GMEM) for 48 h as per the manufacturer's directions. Expression of pol and pol fragment 4 was analysed by RT-PCR and flow cytometry using antibody specific for either the polyhistidine-tag or the V5 epitope-tag (Serotec) and then a fluorescently labelled antibody (fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG; DakoCytomation) (data not shown).
Synthetic VISNA POL peptides.
The predicted POL fragment 4 amino acid sequence was used to synthesize 36 overlapping 15-mer peptides (P1.1–1.35: offset by 5 aa, P1.36 offset by 4 aa) by using a 96-well Pepscan method (Cambridge Peptides). Eight overlapping 12-mer peptides, offset by 2 aa (P2.1–2.7) or 1 aa (P2.8), corresponding to POL amino acid positions 612–636 were synthesized by Mimotopes (Fig. 1). Peptides were dissolved in sterile 0.1 M HEPES pH 7.4 in 40 % acetonitrile to make 5 mg ml–1 stocks (this was assumed for the pepscan peptides from the usual pepscan yield) and stored at –80 °C. The peptide stocks were diluted in RPMI 1640 containing 2 mM L-glutamine, 20 mM HEPES, 100 U penicillin and 100 µg streptomycin ml–1 and 50 µM 2-mercapoethanol with 10 % FCS (10 % RPMI) to a final concentration of 20 µg ml–1 for each peptide before use (pools of peptides were used at 20 µg ml–1 for each peptide).
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) before sheep were infected or immunized. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 100 U penicillin and 100 µg streptomycin ml–1 and 10 % FCS (10 % DMEM).
Preparation of sheep peripheral blood mononuclear cells (PBMCs) as CTL effectors.
Preparation of CTL effectors from PBMCs was performed as described previously by stimulation for 14 days on autologous VISNA-infected skin cell fibroblasts in the presence of interleukin (IL)-2 (Blacklaws et al., 1994). Viable cells from cultures were adjusted to the required concentrations in 10 % RPMI 1640 for use as effectors in CTL assays.
CTL chromium release assay.
Sheep skin cells, either autologous or heterologous, were used as target cells to present VISNA antigens, as in Blacklaws et al. (1994). For VISNA-infected targets, sheep skin cells in 10 % DMEM were plated into 96-well flat-bottom plates at 1x104 cells per well and cultured overnight, and cells were either mock infected or infected for 24 h with 0.5 TCID50 VISNA EV1 per cell in DMEM supplemented as above but with 2 % FCS (2 % DMEM) then labelled with 1.0 µCi (37 kBq) Na251CrO4 (51Cr) in 2 % DMEM per well overnight, washed four times and used. For rMVA-infected targets or peptide-loaded targets, skin cells were plated into 96-well flat-bottom plates at 1x104 cells per well with 1.0 µCi (37 kBq) 51Cr in 10 % DMEM overnight. rMVA-infected targets were washed three times, then either mock infected or infected with 10 p.f.u. rMVA per cell for 4 h in 2 % DMEM, washed once more and used. Targets loaded with peptides were washed four times and incubated with peptides for 1 h in half the final reaction volume at double the final concentration (20 µg ml–1 10 % RPMI 1640) then effectors were added to give the final reaction volume and peptide at 10 µg ml–1. Triplicates of two dilutions of lymphocyte effectors were added to target cells to give a final volume of 100 µl per well. Effectors and targets were incubated for 8–16 h at 37 °C then 40 µl supernatant was mixed with 120 µl Optiphase Hisafe 3 (Perkin-Elmer) in 96-well ELISA plates and counted by scintillation counting (1450 Microbeta counter; Wallac). Spontaneous and maximal 51Cr release was determined by incubating target cells in medium or 1 % Triton X-100, respectively. Specific chromium release, i.e. lysis of cells was calculated by the formula: % specific lysis=[(experimental release–spontaneous release)/(maximal release–spontaneous release)]x100. The mean±SD of triplicate wells is shown in the graphs and tables.
Inhibition of antigen presentation by MHC class I mAbs.
Anti-MHC class I antibody inhibition of antigen presentation was assessed as follows: skin cell targets were prepared as described above. One hour before the addition of effectors, targets were incubated with anti-ovine or -bovine MHC class I or a non-specific mouse IgG1 mAb to give a final concentration of 10 µg ml–1 in 100 µl after the addition of effectors. Finally, effectors were incubated with targets for 12 h.
Immunization of animals with plasmid DNA and rMVA.
Plasmid DNA of pcDNA3.1polfrag4 and pcDNA3.1pol was prepared using a Plasmid Preparation kit (Sigma-Aldrich) as per the manufacturer's directions. Finally, DNA was quantified using the PicoGreen DNA quantification kit (Invitrogen) as per the manufacturer's instructions. For immunization, DNA/polyethylenimine (PEI, 25 kDa branched; Sigma-Aldrich, cat. no. 40 872-7) complexes were prepared at ratios of 1 : 10 DNA phosphate to PEI nitrogen (i.e. 1 µg DNA : 1.29 µg PEI) in water for injection. The complexes were allowed to stand for at least 20 min before use. Animals were given 400 µg plasmid by injection, half given intramuscularly and half subcutaneously. rMVAs were purified using sucrose density-gradient centrifugation (Madalinski et al., 1977). Animals were immunized with 3x108 p.f.u. rMVA subcutaneously. Animals received injections of DNA twice then rMVA once at 3 weekly intervals. Sheep B1117 and B1123 were immunized with VISNA pol fragment 4; sheep B1127 and B1129 with whole length pol (minus 78 bp at the C-terminal end). PBMCs were tested for precursor CTL activity before immunization, 3 weeks after the second DNA immunization and 3 or 6 weeks after the rMVA boost.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) recognized the pol gene product. The two Friesland sheep used displayed different reactivities with one (J057, Table 1) recognizing the gag gene product (p55) weakly, specifically the p17 N-terminal matrix region of this gene, as well as recognizing the env gene product strongly. The other Friesland sheep (J141, Table 1) showed reactivity to both the env and pol gene products. The two Lleyn sheep also displayed different reactivities with one (C139, Table 1, note the data shown is at an effector : target ratio of 50 : 1 to show weak reactivity) recognizing the gag gene product (p55), this time specifically in the p25 central capsid region of the gene, the env gene product weakly, and the rev and vif gene products. The other Friesland sheep (C175, Table 1) showed poor CTL reactivity to VISNA-infected cells and this was specific for the env gene product. Except for the tat gene product we have therefore shown that all the gene products of VISNA may act as CTL targets in sheep.
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Mapping CTL epitopes in VISNA POL antigen
In strain EV1, the pol gene is 3267 bp long and encodes an 1088 aa protein. To define a small region of the antigen for peptide mapping, six rMVA viruses expressing overlapping POL antigen fragments (sizes ranged from 188 to 230 aa and fragments overlapped by 20–25 aa) were used to infect targets in CTL assays. Although background values were relatively high (
30 % lysis), MVApolfragment4-infected autologous cells showed similar levels of specific lysis to those of MVApol-infected autologous cells with both sheep effectors (Fig. 2a, b). Other rMVA pol fragment (MVApolfragment1–3 and 5–6)-infected cells showed background levels of lysis. These levels of lysis were as high as lysis of target cells infected by VISNA itself (data not shown).
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). This indicated that peptide pool 3 contained a peptide(s) presented to CTL from sheep B1125 and B1139. Skin cell targets from sheep B1125 were also able to present peptide pool 3 peptides to CTL effectors from sheep B1139 with levels of target cell lysis similar to those in CTL assays with autologous CTL effectors and targets (Fig. 3).
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). To map the CTL epitope(s) in this 25 aa region of VISNA POL, eight overlapping 12 aa peptides, offset by 2 aa (P2.1–2.7) or 1 aa (P2.8) were synthesized (Fig. 1a) and individually loaded into targets for CTL assays. The longest peptides isolated from cattle MHC class I are 12 aa in length (Gaddum et al., 1996). Four peptides, P2.1 (DSRYAFEFMIRN), P2.5 (MIRNWDEEVIKN), P2.7 (WDEEVIKNPIQA) and P2.8 (DEEVIKNPIQAR) induced the highest and similar levels of lysis of targets by CTL (Fig. 4c, d). Peptides P2.2, P2.4 and P2.6 caused approximately half the level of lysis by CTL as P2.1, P2.5, P2.7 and P2.8 (Fig. 4c, d). Peptide P2.3 was not presented to the CTL effectors used. None of the peptides sensitized heterologous target cells to lysis by sheep CTL (Fig. 4). The C-terminal 15-mer peptide from POL, P1.36, was used as a control peptide on targets and did not sensitize target cells to lysis (Fig. 4c, d). Comparison of the four most highly active 12-mer peptides showed that P2.1 and P2.5 shared 4 aa, P2.5 and P2.7 shared 8 aa, P2.5 and P2.8 shared 7 aa, and P2.7 and P2.8 shared 11 aa. This, with the pattern of recognition of the intervening peptides (especially peptide P2.6, which was not presented efficiently), suggests that a cluster of three or more epitopes are present in this 25 aa region of POL: within DSRYAFEFMIRN (612–623), MIRNWDEEVIKN (620–631) and DEEVIKNPIQA (625–635) (Fig. 1a).
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). Peptide concentrations from 0.01 to 10 µg ml–1 (6.5–6500 nM) sensitized target cells for lysis by CTL. They were not effective at 1 ng ml–1 (0.65 nM) when levels of lysis were similar to those for control peptide (P1.36)-loaded targets (Fig. 5).
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). Consistent results were obtained using effectors from sheep B1125 (data not shown). The CTL epitopes in POL (recognized by B1125 and B1139) were therefore presented to CTL by MHC class I molecules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Blacklaws et al., 1994; Griffin et al., 1978; Larsen et al., 1982; Reyburn et al., 1992; Sihvonen, 1981; Torsteinsdóttir et al., 1992). Fibroblasts from individual sheep were able to present different VISNA antigens to CTL, probably because of varying MHC class I backgrounds. TAT was the only protein that did not contain a CTL epitope. VISNA tat has recently been compared with HIV-1 vpr because of the lack of a TAR element in the VISNA LTR and weak upregulation of viral promoter activity in the presence of TAT (Villet et al., 2003). HIV VPR and TAT both contain CTL epitopes. With only six sheep used here it is likely that VISNA TAT will also prove to contain epitopes when more sheep are studied rather than there being an inherent lack of processing or immunogenicity of this protein. With bluetongue virus, it has been found that there is considerable diversity in the CTL recognition patterns in sheep tested (Janardhana et al., 1999). The specific lysis of targets was variable in some of the different experiments shown here. This variability was independent of the animal analysed, or the types (infected or peptide loaded) of targets used in experiments and we could not identify the cause. The majority of assays were incubated for 16 h, although in attempts to reduce background levels, assays of less than 16 h were also performed. These reduced overall specific lysis as well as background levels. We used the same sources of medium, FCS and IL-2 throughout. We also kept skin cell targets between passage 6 and 20, although we have not noted differences in the ability to kill different ages of skin cells before. One possible explanation we have is that the infection of the fibroblast feeders used to activate CTL cultures may have varied with different stocks of VISNA. VISNA causes a slow spreading infection of fibroblasts throughout the culture period and if this varied it may have affected the activity of the CTL generated.
Cytotoxic activity may be mediated by CD8+ CTL, CD4+ CTL or NK cells. However, it has been shown that a major proportion of cell-mediated cytotoxicity is performed by MHC class I-restricted CD8+ T cells in different viral infections (Harty et al., 2000). Using the culture method employed here or macrophages as antigen presenting cells, it has been shown previously that VISNA-specific CTL activity is mediated by CD8+ T lymphocytes (Blacklaws et al., 1994; Lee et al., 1994). POL (expressed by MVA or as peptides)-specific cytotoxicity reported here was inhibited by mAb specific for MHC class I antigen. There was also no evidence that the sheep skin cells used as targets express MHC class II (by flow cytometry, data not shown). This indicates that VISNA POL epitopes were presented in a MHC class I-restricted manner to CTL.
In a group of highly related sheep sharing MHC class I antigens, several epitopes within POL were shown to cluster in a short 25 aa region of the protein associated with RNaseH activity. We have not mapped these epitopes to presentation by particular class I molecules and so they may be presented by different class I molecules. The first amino acid (D) of this region is one of the conserved aspartic acid residues necessary for RNaseH activity in Escherichia coli (Kanaya et al., 1990, Fig. 1) and three of the first 4 aa are conserved amongst all the lentiviruses. This 25 aa region of POL is well conserved within the small ruminant lentiviruses, with common substitutions being conservative (Fig. 1b).
CTL epitopes have been mapped in all lentiviruses except CAEV and BIV. HIV and SIV have the largest epitope database (www.hiv.lanl.gov/content/index) where epitopes are also known in the context of different MHC class I-presenting molecules. Epitopes are often clustered on protein sequences in conserved regions and this is thought to be caused by natural antigen processing on the proteasome, presentation and the ability to screen cross-reacting peptide sequences within individuals (Lucchiari-Hartz et al., 2003; Walker & Korber, 2001). There is limited knowledge of EIAV epitopes but a cluster of epitopes is present in the GAG matrix and capsid regions (Chung et al., 2004). There is also evidence of epitope clustering in other virus families, e.g. hepatitis B virus (Chisari & Ferrari, 1995) and hepatitis C virus (Rehermann, 2000; Wong et al., 2001). Therefore, the presence of a cluster of epitopes in a conserved region of a protein as we see here is characteristic of antiviral responses in diverse species.
We have not defined optimal length peptides here but have used 12-mer peptides. One optimal 9-mer (ISIDQILEA) epitope has been defined in a conserved region of bovine leukaemia virus (BLV) gp51 in merino sheep (Hislop et al., 1998). Several optimal epitope peptides have been defined with corresponding BoLA presentation molecules in cattle. These vary from 9 to 12-mer peptides with 9 or 11-mer peptides being most commonly seen (Gaddum et al., 1996; Graham et al., 2008; Sinnathamby et al., 2004). Similarly, the length of CD8+ CTL epitopes in humans varies from 8 to 11-mers depending on the specific MHC class I allele-binding groove. Thus, 12-mer peptides gave a reasonable chance of containing epitopes for MHC class I presentation in sheep, although the sensitivity of the assay may have been reduced by using suboptimal peptide lengths.
Peptides that caused sensitization of targets to CTL were active to 6.5 nM. EIAV epitopes for CTL in gag have been classified as having high functional avidity if optimal peptides sensitize targets at <11 nM or low functional avidity if >1000 nM is required, with reactivity to a high-functional-avidity epitope correlating with increased control of virus [more days since the last clinical episode; Chung et al. (2005)]. As EIAV is macrophage-tropic this is particularly relevant for VISNA. The peptides used here were not optimal and their binding range was of high avidity, suggesting they may be of functional use in viral control.
By CD8+ T-cell depletion and an acute infection model, we have not been able to show a role for CTL in control of VISNA infection (Eriksson et al., 1999); however, the slow rate of virus replication and residual CD8+ T-lymphocyte populations in tissues during these experiments may mean that there was sufficient CTL activity remaining to control VISNA replication. The VISNA-infected sheep used here to map the CTL epitopes in POL had no clinical signs of VISNA infection during these experiments or at post-mortem, suggesting they were controlling virus replication. It is in the late stages of VISNA disease that increased virus load and antigen are seen (Brodie et al., 1992; McNeilly et al., 2007; Narayan & Clements, 1989). With immunodeficiency viruses, control of virus load has been linked to the quality and strength of the antiviral CTL response (Heeney & Plotkin, 2006).
The results of CTL assays with PBMCs isolated from DNA-prime–rMVA-boost immunized sheep indicated that subunit vaccines were capable of inducing the same anti-VISNA CTL specificity as in VISNA-infected sheep. There was weak CTL activity induced after DNA priming in a subset of sheep (data not shown) and this reactivity increased with the MVA boost. This is the first study to show induction of CTL by a prime-boost method in sheep. It is likely that the full-length pol gene and pol fragment 4 constructs also contained T helper (MHC class-II-presented) epitopes, which may have induced CD4+ T-lymphocyte responses, which then help in the induction of the CD8+ lymphocyte responses. It will be interesting to try induction of CTL reactivity by minimal epitopes with this method.
Advantages of DNA-prime and recombinant virus-boost immunization strategies are well recognized. A popular prime-boost regimen is that of DNA priming followed by recombinant poxvirus or adenovirus boosting (Amara et al., 2002; McDermott et al., 2005; Wang et al., 2005). Such prime-boost regimens are able to raise cellular immunity, and this has produced encouraging results with the long-sought malaria vaccine (Gilbert et al., 2006; McConkey et al., 2003) and in models of HIV infection (Heeney & Plotkin, 2006). The levels of specific CTL activity in immunized sheep may be improved further if optimal immunization dose, adjuvant, route and interval are investigated (Babiuk et al., 2003; Chaplin et al., 1999; De Rose et al., 2002; Gurunathan et al., 2000; Scheerlinck et al., 2004; Watkins et al., 2005). Immunization to induce CTL activity can provide protection from retrovirus infection in sheep: immunization with an optimal CTL peptide within BLV gp51 protected sheep from challenge with BLV; however, this protection was not total as there was late isolation of BLV from immunized sheep (Mateo et al., 2001).
In conclusion, we describe the first, to our knowledge, systematic screening for CD8+ CTL epitopes in VISNA antigens. Results showed that all antigens of VISNA acted as CTL targets except for TAT in this study using a limited number of sheep. POL antigen was recognized by CTL from sheep sharing MHC class I alleles, and one cluster of at least three CTL epitopes was identified. Immunization by DNA-prime-boost using rMVA induced specific CTL and this will allow further investigation of the role of CD8+ T lymphocytes in control of this macrophage- and dendritic-cell-tropic lentivirus infection.
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ACKNOWLEDGEMENTS |
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Received 1 April 2008;
accepted 9 June 2008.
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