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1 Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK
2 Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK
3 Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France
4 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
Correspondence
Julia H. Kydd
julia.kydd{at}aht.org.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Edington et al., 1994; Slater et al., 1994; Welch et al., 1992). It is responsible for substantial economic losses within the bloodstock industry and in addition has serious welfare implications. To control the disease, it will be necessary to reduce nasopharyngeal virus shedding and cell-associated viraemia, and this reduction is likely to be achieved by the stimulation of virus-neutralizing antibody and cytotoxic T lymphocytes (CTL) (Allen, 2002; Allen et al., 1999). While current vaccines containing inactivated virus stimulate virus-neutralizing antibody (Heldens et al., 2001) and reduce the amount and duration of infectious virus, which is shed from the nasopharynx (Hannant et al., 1993; Heldens et al., 2001), they fail to prevent infection. Sporadic outbreaks of EHV-1-related abortion and neurological disease therefore continue to occur (Patel & Heldens, 2005). These outbreaks are likely caused by failure of the available vaccines to stimulate CTL and thereby control cell-associated viraemia.
EHV-1-specific CTL can recognize and lyse virus-infected cells in a genetically restricted, antigen-specific manner (Allen et al., 1995) and are important in control of infection. Ponies with high pre-infection frequencies of CTL, as measured by limiting dilution analysis, show a reduction in clinical and virological signs of disease, including abortion (Kydd et al., 2003; O'Neill et al., 1999). Therefore the stimulation of CTL by vaccination is an important aim. However, EHV-1 has a large genome containing 76 open reading frames. Some EHV-1 proteins and/or transcripts have potentially deleterious effects in vitro such as immunosuppression (Charan et al., 1997; Hannant et al., 1999) and downregulation of MHC class I molecules (Ambagala et al., 2004; Rappocciolo et al., 2003). Therefore the identification of CTL-target proteins and their inclusion in vaccines that present antigen by the endogenous route are essential for improved vaccination strategies (Minke et al., 2004). Early data suggest that CTL-target proteins are encoded by genes of the immediate early (IE) and early classes of EHV-1 (Allen et al., 1995). Recently, Soboll et al. (2003) have shown that the single IE gene of EHV-1 (gene 64) contains CTL epitopes that are presented by an MHC class I allele or alleles carried on the serologically defined A3 haplotype. Their approach involved the biolistic transfection of dendritic cells with plasmids encoding individual EHV-1 gene products. These transfected dendritic cells were then used to induce effector CTL from primed ponies, which were then tested against whole virus-infected target cells. However, the allele(s) responsible for presentation of the EHV-1 peptide(s) encoded by gene 64 was not identified in that study. In contrast, the current paper takes a molecular approach, enabling identification of EHV-1 proteins that are recognized by CTL and the equine MHC class I genes responsible for their presentation. In particular, it is important to identify the MHC class I alleles involved in the presentation of gene 64 epitopes, to characterize further their interactions and enable construction of MHC class I–peptide tetramers to characterize immune responses ex vivo.
The MHC of the horse is known by its species-specific designation as the equine leukocyte antigen (ELA-A) region. Serological reagents identify 17 internationally accepted ELA specificities that correspond to haplotypes (Antczak, 1992; Antczak et al., 1982; Bailey et al., 2000). However, the serological reagents fail to distinguish the different gene products expressed on these haplotypes or subgroups of the haplotypes. The first physical map of the equine MHC has been defined through a contig of bacterial artificial chromosome (BAC) clones produced using DNA from a horse homozygous for the ELA-A3 serological haplotype (Gustafson et al., 2003). Recently, the genomic sequences of 15 horse MHC class I genes and pseudogenes were obtained from these BAC clones (Tallmadge et al., 2005). Seven of these genes are transcribed and predicted to be expressed. These include four genes that have the characteristics of classical MHC class I genes (designated 3.1, 3.2, 3.3 and 3.4, where ‘3’ indicates the haplotype of origin and the number of a fully sequenced gene) and three so-called non-classical class I genes (3.5, 3.6 and 3.7). Four of these seven genes have been described previously from cDNA clones (Ellis et al., 1995). B2 (classical gene), A1, C1 and E1 (all non-classical genes) of Ellis et al. (1995) correspond to 3.1, 3.5, 3.6 and 3.7, respectively (Tallmadge et al., 2005). The present study focused on the classical B2 gene (equivalent to 3.1) and the C1 non-classical gene (equivalent to 3.5), which are expressed by the ELA-A3 haplotype. The ELA-A7 haplotype, which expresses classical B1 and B4 genes but not the C1 non-classical gene, was used as a control. This model was studied because the B2 and C1 genes have been cloned and expressed (Ellis et al., 1995), and the ELA-A3 haplotype (as defined serologically) is expressed by a large proportion of the thoroughbred population (Bodo et al., 1994), which forms a major market for vaccines. The ELA-A3 serological specificity is apparently heterogeneous, however, since effector CTL from ponies typed as subgroup A3.1 failed to lyse targets from a pony typed as subgroup A3.2 (Soboll et al., 2003).
The work described in this paper used a molecular approach to screen a selection of EHV-1 genes in order to identify the CTL-target proteins of EHV-1 presented by the MHC class I B2 molecule. It also examined the role of the C1 gene. The data derived from these studies are an essential prerequisite for the identification of EHV-1 target peptides and ultimately the synthesis of novel reagents such as MHC–peptide tetramers to characterize immune responses ex vivo. The technique is also applicable to other equine viruses for which CTL-target proteins and their presenting MHC alleles are unknown.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) into the pcDNA3 mammalian expression vector (Invitrogen). Each gene was stably transfected by electroporation into the murine mastocytoma P815 cell line (H-2d; European Cell and Culture Collection, Centre for Applied Microbiology and Research, Porton Down, Wiltshire), and expression was confirmed by using indirect immunofluorescence and the anti-horse MHC class I monoclonal antibody (mAb) CZ3. A total of eight EHV-1 genes were chosen for screening as CTL targets (Table 1). Seven of these were amplified from DNA derived from EHV-1 strain Ab4/13, a plaque-purified clone of wild-type strain Ab4/8 (Telford et al., 1992) that was isolated in the UK. Primers were designed from the published sequences to include restriction enzyme sites (Table 1) at the 5' and 3' ends, and the amplicon cloned into pcDNA6/V5-His (V5 tag at C terminus; Invitrogen). A plasmid encoding open reading frame (ORF) 16 (glycoprotein C; a kind gift from Professor Hartwig Huemer, Institute of Hygiene, University of Innsbruck, Austria) was subcloned into pcDNA6 (Invitrogen). All constructs were sequenced prior to their further use. A panel of stable P815 transfectants expressing the class I allele B2 and each of the eight EHV-1 genes was generated using electroporation, limiting dilution cloning and selection with G418 (Calbiochem), and blasticidin S (Invitrogen). In addition ORFs 12, 14 and 64 were transfected into P815 cells expressing the class I allele C1. Expression of ORF 16 was confirmed using the murine mAbs 8F8 and 7F11 specific for gC (Sinclair et al., 1989) and indirect immunofluorescence. Expression of the other seven EHV-1 genes was confirmed by Western blot analysis using a mAb to the V5 tag (Invitrogen).
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). Group 1 comprised four Welsh Mountain ponies, while group 2 consisted of four thoroughbred horses (Table 2). The ponies were 6C31 (A3/x), 302A (A7/A7), 6132 (A3/A3) and her son 3862 (A3/A3). The ELA-A3 haplotype encodes the classical class I gene B2, several non-classical genes including C1, and additional classical class I genes (Ellis et al., 1995; Tallmadge et al., 2005). The ELA-A7 haplotype expresses the classical MHC class I genes B1 and B4 but not the non-classical C1 gene (Ellis et al., 1995). Expression of the B2 and C1 genes of the ELA-A3 haplotype and the B1 and B4 genes of the ELA-A7 haplotype was confirmed in group 1 ponies by PCR amplification and sequencing of expressed class I genes (Ellis et al., 1995). Ponies 6C31, 302A and 3862 were primed to EHV-1 by intranasal experimental infections with strain Ab4/15 (a plaque-purified clone of the virulent Ab4/8 strain derived from a field outbreak in the UK; Telford et al., 1992) as described previously (Mumford, 1994). Pony 6132 had experienced only field infections with EHV-1.
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101.6) of EHV-1/-4 cross-reactive complement-fixing antibodies (Thomson et al., 1976). Mares AM and FW were also seropositive for EHV-1 glycoprotein G as measured by ELISA (Svanova Biotech) at the time of blood collection. Mare FW (ELA-A5/A5) donated blood for heterologous target cells only. PBMC from the Cornell mares were transported in complete RPMI [RPMI 1640 supplemented with 10 % (v/v) fetal calf serum, 2 mM L-glutamine, 100 U penicillin ml–1, 100 µg streptomycin ml–1; all ingredients from Sigma] to the Animal Health Trust (AHT) (UK) by air for testing.
PBMC were enriched from heparinised blood by centrifugation over Ficoll (Pharmacia), washed in PBS and viable cells were counted and assessed for viability by trypan blue dye exclusion. Cells from the AHT ponies and Cornell horses were used fresh, stored in complete RPMI for up to 2 days at 4 °C or cryopreserved (O'Neill et al., 1999) for later use in the CTL assay. All research studies involving animals were approved by the Ethical Review Committee of the AHT and complied with the Animals (Scientific Procedures) Act 1986.
CTL assay.
A 4 h chromium release assay was used to detect EHV-1-specific, MHC class I-restricted CTL activity (Allen et al., 1995) that is mediated by CD8+ lymphocytes. The previously validated assay was modified slightly by using EHV-1 strain Ab4/15, a plaque-purified clone (Telford et al., 1992) to induce effectors and to infect target cells. Transfected P815 cells were also used as target cells. These were cultured in complete RPMI and split the day before radioisotope labelling. Effector CTL-to-target cell ratios ranged from 100 : 1 to 12 : 1 with three replicates each. Percentage spontaneous release was 
25 % in all experiments. Percentage specific lysis was calculated according to the standard formula:
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In some experiments, the assay was adapted to determine the effects of several parameters on the percentage specific lysis. These included infection of target cells or induction of effectors with modified vaccinia virus (NYVAC) constructs; these are described in detail below. The results reported are each representative of at least three separate experiments and are expressed as the mean±standard deviation.
Origin of NYVAC constructs.
The recombinant virus constructs (vP1014 and vP884) were generated by the insertion of the IE gene 64 or ORF 33 (encoding glycoprotein B), respectively, of EHV-1 (originating from the Kentucky D strain) into the highly attenuated vaccinia-derived New York (NYVAC) strain (Piccini et al., 1987). These recombinant viruses were developed by Virogenetics according to standard in vitro recombination techniques (Tartaglia et al., 1992). NYVAC is a registered trademark in the United States of Connaught Technology Corporation.
Infection of target cells with NYVAC constructs.
Lymphoblast target cells were infected with either NYVAC gene 64 or NYVAC gB at an m.o.i. of 10 for 1.5 to 2 h prior to labelling with radioisotope and used in the CTL assay.
Induction of effector CTL with NYVAC constructs.
To test the ability of EHV-1 gene 64 to induce CTL in vitro, PBMC from ponies primed by previous experimental infection to EHV-1 strain Ab4/13 were incubated with either NYVAC gene 64 or NYVAC gB at an m.o.i. of 0.3 or 0.6 for 6 or 7 days. Effector CTL were then tested against EHV-1-infected lymphoblasts or transfected P815 cells.
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RESULTS |
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) and co-transfected into P815 cells that expressed the equine MHC class I B2 gene. Plasmids containing ORFs, 12, 14, 16 and 64 were also co-transfected individually into P815 expressing the non-classical C1 gene. Control P815 cells transfected with a single gene encoding either B2, C1 or EHV-1 gene 64 were also produced. These stably transfected cells were used as targets in the CTL assay to identify target proteins.
Induction of virus-specific, genetically restricted effector CTL activity from PBMC
The effector CTL activity induced from PBMC of EHV-1-primed, MHC-typed ponies and thoroughbreds was tested against autologous or heterologous virus-infected lymphoblasts. As shown in Fig. 1, effector CTL induced with EHV-1 from pony 6C31 (ELA-A3/x), pony 302A (ELA-A7/A7) and horse AM (ELA-A3/A3) killed autologous virus-infected targets. The killing was genetically restricted; no mare lysed heterologous, virus-infected target cells. Also, the killing was virus specific; mock-infected lymphoblast targets were not lysed by any mare. These results confirmed the presence of lytic activity by effector CTL that were subsequently used against P815 transfectant target cells. Levels of EHV-1-specific CTL activity were high in all experimentally infected ponies and thoroughbreds throughout the sampling period. CTL stimulated from ponies 6132 and 3862 and thoroughbreds BT and CP (all ELA-A3 homozygotes) showed an identical pattern of genetic restriction to 6C31 (data not shown). The percentage specific lysis against virus-infected, homologous targets at 100 : 1 effector-to-target ratio was 63.0±3 % for pony 3862, 24.0±4 % for pony 6132, 29.3±2 % for horse BT and 44.3±1.9 % for horse CP.
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, EHV-1-specific, effector CTL from pony 6C31 recognized only one of the eight EHV-1 genes screened, when presented by the MHC class I B2 molecule. Lytic activity against double transfectants that expressed the B2 gene and an EHV-1 gene (ORFs 2, 12, 14, 16, 35, 63, 69) was <10 % at 100 : 1 effector-to-target ratio (Table 1). In contrast, the protein encoded by EHV-1 gene 64 was identified as a target antigen for MHC class I B2-specific effector CTL from pony 6C31 (ELA-A3/x) as shown by the lysis of double transfectants (Fig. 2a). The same effector CTL failed to lyse single transfectants expressing only the MHC class I gene B2 or the EHV-1 gene 64. Similar data were obtained from the other two ELA-B2+ ponies 3862 and 6132 (data not shown) and thoroughbred mare AM (Fig. 2b). In contrast, effector CTL from the pony mare 302A (ELA-A7/A7), which does not carry the B2 gene, failed to lyse B2+/gene 64+ double transfectants (Fig. 2c). Transfected target cells expressing the non-classical C1 gene and EHV-1 gene 64 were not lysed by effector CTL from either pony 6C31 [ELA-A3/x (B2+)] or thoroughbred AM [ELA-A3/A3, (B2+)]. Thus, there was T-cell recognition of targets expressing the MHC class I B2 gene and the EHV-1 gene 64, but not targets expressing either the C1 gene and genes 12, 14, 16 and 64, or the B2 gene and genes 2, 12, 14, 16, 35, 63 and 69.
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). Killing of NYVAC gB or mock-infected homologous cells was low and lysis of heterologous cells infected with NYVAC gB or NYVAC gene 64 target cells was <10.9 % (data not shown). Second, NYVAC gene 64 was used to induce effector CTL from lymphocytes of pony 6C31 [ELA-A3/x; (B2+)]. As shown in Fig. 4(a), induction with NYVAC gene 64 stimulated effector CTL, which lysed both whole virus-infected autologous lymphoblasts and B2+/gene 64+ double transfectants. No lysis was detectable when either single transfectants (Fig. 4a) or mock-infected lymphoblasts (data not shown) were used. In contrast, induction with NYVAC gB resulted in no detectable lysis of the same target cells (Fig. 4b). The results confirmed that NYVAC gene 64 derived from Kentucky D strain induced CTL in vitro from ponies primed to EHV-1 by infection with strain Ab4/13 and lysed target cells infected with Ab4/13. Similarly, NYVAC gene 64-infected target cells were lysed by effector CTL from a B2+ pony primed in vivo to EHV-1 by experimental infection with strain Ab4/13. These data also demonstrate that at least for gene 64, strains of EHV-1 derived from North American or European isolates can stimulate cross-reactive CTL in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Soboll and colleagues used dendritic cells transfected with plasmids encoding EHV-1 genes to induce CTL from EHV-1-primed, MHC class I-typed ponies. Effector CTL from ponies with the ELA-A3 haplotype, which had been induced with gene 64, lysed whole virus-infected target cells. In contrast, using a murine mastocytoma cell line (P815) transfected with MHC class I and EHV-1 genes, we have demonstrated that the MHC class I gene B2 presents peptide(s) encoded by gene 64 to ELA-A3 effector CTL derived from EHV-1-primed ponies and thoroughbred horses. Our work therefore, for the first time, identifies B2 as an MHC class I gene responsible for the genetic restriction of this viral gene product.
Equine MHC class I haplotypes are complex and the serological typing methods currently employed have limitations. These limitations have been shown by differences in CTL recognition between animals with the ELA-A3 (Soboll et al., 2003) and ELA-A5 (Zhang et al., 1998) serological haplotypes. For example, the anti-ELA-A3 antisera panel divided ponies into two subgroups, namely 3.1 and 3.2. EHV-1-specific CTL generated from subgroup 3.1 failed to lyse virus-infected targets from subgroup 3.2 (Soboll et al., 2003). Thus, further molecular analysis of the ELA-A3 haplotype in thoroughbreds should be performed on a representative sample to ensure that the haplotype has the same composition in each case and to ensure the consistent presence of the B2 allele. More generally, sequencing of the equine MHC is needed to develop more sophisticated typing methods. Encouragingly, the MHC class I genes from an animal homozygous for a serologically defined ELA-A3 haplotype have recently been sequenced (Tallmadge et al., 2005), which confirmed the presence of the ELA-B2 allele but also revealed three further putative classical class I genes that were shown to be transcribed. Earlier data (Ellis et al., 1995) suggest that most horse MHC class I haplotypes transcribe and express no more than two genes at easily detectable levels. Furthermore B2 is expressed at a high level in our A3+ ponies and is transcribed at a significantly higher level than any other class I gene (Ellis et al., 1995). Thus, in addition to B2, it is possible that at least one of potentially three other ELA-A3 classical alleles may also present EHV-1 viral peptides, which may or may not be encoded by gene 64. Further work is clearly required to determine the functional relevance of the additional MHC class I genes and to determine if most or all haplotypes express four classical class I genes. It is likely that even within the ELA-A3 haplotype, further CTL-target proteins may require identification. Determination of CTL-target proteins for MHC class I alleles expressed by other major thoroughbred haplotypes, e.g. ELA-A2, -5 and -A9, would allow use of recombinant vaccines that could stimulate CTL responses in >95 % of the thoroughbred population. The T-cell receptor repertoire may also differ between ELA-A3+ animals and this may affect the CTL response.
The NYVAC gene 64 construct proved useful in confirming that this antigen was genetically restricted by alleles expressed by the ELA-A3 haplotype, including the B2 gene. It also provided evidence of cross-reactivity of CTL responses stimulated by different strains of EHV-1, namely the Ab4/15 (isolated in the UK), Kentucky D (USA) and a North American field strain, suggesting that the immunogenic peptides are sufficiently similar for CTL recognition. This conclusion is substantiated by the high degree of conservation in the nucleotide sequences of Ab4/13 (Telford et al., 1992), KyA (Grundy et al., 1989) and KyD (Tartaglia et al., 1992), with only four base changes in gene 64, of which two result in an amino acid substitution at positions 1073 and 3700. If similar conservation is present in the strains of EHV-1 circulating in the field, CTL are likely to be cross-reactive and this is reassuring for future vaccine development in an international market. In addition, this construct may be used to screen animals of known MHC class I haplotypes in vitro for their response to gene 64. Recently, we demonstrated that vaccination of ponies that express the A3 haplotype with NYVAC gene 64, resulted in the stimulation of virus-specific effector CTL and interferon-gamma synthesis (Paillot et al., 2005, 2006). The product of gene 64 is therefore a strong candidate for inclusion in future vaccines designed to induce cellular immune responses, at least in the B2+ subpopulation of horses. Used in conjunction with other vaccines (e.g. either inactivated virus or glycoprotein subunits), which contain antibody epitopes, and administered intramuscularly and intranasally, there is a realistic chance that this vaccination strategy will improve protection.
In conclusion, we have identified the MHC class I molecule encoded by the B2 gene as being capable of presenting peptide from EHV-1 gene 64 epitopes to effector CTL. The system is applicable to other viruses and will enable the identification of CTL-target proteins and their genetic restricting elements. For EHV-1, the characterization of further CTL-target proteins and their restricting MHC class I alleles is dependent on the identification and cloning of additional equine MHC class I alleles and the application of molecular typing methods (Chung et al., 2003). Immediate future work will focus on the identification of the peptide(s) encoded by gene 64, which are presented by the B2 molecule. This will ultimately enable the construction of MHC class I–virus peptide tetramers as has been achieved successfully with Equine infectious anemia virus (Mealey et al., 2005) and many other pathogens (Klenerman et al., 2002). Further dissection of the in vivo immune response to EHV-1 using tetramers will yield valuable epidemiological information and ultimately lead to improved vaccination strategies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Allen, G., Yeargan, M., Costa, L. R. & Cross, R. (1995). Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1. J Virol 69, 606–612.[Abstract]
Allen, G. P., Kydd, J. H., Slater, J. & Smith, K. C. (1999). Advances in understanding of the pathogenesis, epidemiology and immunological control of equine herpesvirus abortion. In Equine Infectious Diseases, vol. VIII, pp. 129–146. Cambridge, UK: R&W Publications (Newmarket) Ltd.
Ambagala, A. P. N., Gopinath, R. S. & Srikumaran, S. (2004). Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1. J Gen Virol 85, 349–353.
Antczak, D. F. (1992). The major histocompatibility complex of the horse. In Equine Infectious Diseases, vol. VI. Cambridge, UK: R&W Publications (Newmarket) Ltd.
Antczak, D. F., Bright, S. M., Remick, L. H. & Bauman, B. E. (1982). Lymphocyte alloantigens of the horse. I. Serologic and genetic studies. Tissue Antigens 20, 172–187.[Medline]
Antczak, D. F., Bailey, E., Barger, B. & 7 other authors (1986). Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25–27 April 1984. Anim Genet 17, 363–373.[Medline]
Bailey, E., Marti, E., Fraxer, D. G., Antczak, D. F. & Lazary, S. (2000). Immunogenetics of the horse. In The Genetics of the Horse, pp. 123–155. Edited by A. T. Bowling & A. Ruvinsky. Wallingford, UK: CABI Publishing.
Bodo, G., Marti, E., Gaillard, C., Weiss, M., Bruckner, L., Gerber, H. & Lazary, S. (1994). Association of the immune response with the major histocompatibility complex in the horse. In Equine Infectious Diseases, vol, VII, pp. 143–151. Edited by H. Nakajima & W. Plowright. Newmarket, UK: R&W Publications (Newmarket) Ltd.
Charan, S., Palmer, K., Chester, P., Mire-Sluis, A. R., Meager, A. & Edington, N. (1997). Transforming growth factor-beta induced by live or ultraviolet-inactivated equid herpes virus type-1 mediates immunosuppression in the horse. Immunology 90, 586–591.[CrossRef][Medline]
Chung, C., Leib, S. R., Fraser, D. G., Ellis, S. A. & McGuire, T. C. (2003). Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products. Eur J Immunogenet 30, 387–396.[CrossRef][Medline]
Edington, N., Welch, H. M. & Griffiths, L. (1994). The prevalence of latent equid herpesviruses in the tissues of 40 abattoir horses. Equine Vet J 26, 140–142.[Medline]
Ellis, S. A., Martin, A. J., Holmes, E. C. & Morrison, W. I. (1995). At least four MHC class I genes are transcribed in the horse: phylogenetic analysis suggests an unusual evolutionary history for the MHC in this species. Eur J Immunogenet 22, 249–260.[Medline]
Grundy, F. J., Baumann, R. P. & O'Callaghan, D. J. (1989). DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene. Virology 172, 223–236.[CrossRef][Medline]
Gustafson, A. L., Tallmadge, R. L., Ramlachan, N., Miller, D., Bird, H., Antczak, D. F., Raudsepp, T., Chowdhary, B. P. & Skow, L. C. (2003). An ordered BAC contig map of the equine major histocompatibility complex. Cytogenet Genome Res 102, 189–195.[CrossRef][Medline]
Hannant, D., Jessett, D., O'Neill, T., Dolby, C. A., Cook, R. F. & Mumford, J. A. (1993). Responses of ponies to equid herpesvirus-1 ISCOM vaccination and challenge with virus to the homologous strain. Res Vet Sci 54, 299–305.[Medline]
Hannant, D., O'Neill, T., Ostlund, E. N., Kydd, J. H., Hopkin, P. J. & Mumford, J. A. (1999). Equid herpesvirus-induced immunosuppression is associated with lymphoid cells and not soluble circulating factors. Viral Immunol 12, 313–321.[Medline]
Heldens, J. G. M., Hannant, D., Cullinane, A. A., Prendergast, M. J., Mumford, J. A., Nelly, M., Kydd, J. H., Weststrate, M. W. & van den Hoven, R. (2001). Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4). Vaccination/challenge experiments in foals and pregnant mares. Vaccine 19, 4307–4317.[CrossRef][Medline]
Huemer, H. P., Nowotny, N., Crabb, B. S., Meyer, H. & Hubert, P. H. (1995). gp13(EHV-gC): a complement receptor induced by equine herpesviruses. Virus Res 37, 113–126.[CrossRef][Medline]
Klenerman, P., Cerundolo, V. & Dunbar, P. R. (2002). Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol 2, 263–272.[CrossRef][Medline]
Kydd, J. H., Wattrang, E. & Hannant, D. (2003). Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes correlate with protection against abortion following experimental infection of pregnant mares. Vet Immunol Immunopathol 96, 207–217.[CrossRef][Medline]
Mealey, R. H., Sharif, A., Ellis, S. A., Littke, M. H., Leib, S. R. & McGuire, T. C. (2005). Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes. Virology 339, 110–126.[CrossRef][Medline]
Minke, J. M., Audonnet, J. C. & Fischer, L. (2004). Equine viral vaccines: the past, present and future. Vet Res 35, 425–443.[CrossRef][Medline]
Mumford, J. A. (1994). Abortigenic and neurological disease caused by experimental infection with equid herpesvirus-1. In Equine Infectious Diseases, vol. VII, pp. 261–276. Edited by H. Nakajima & W. Plowright. Cambridge, UK: R&W Publications (Newmarket) Ltd.
O'Neill, T., Kydd, J. H., Allen, G. P., Wattrang, E., Mumford, J. A. & Hannant, D. (1999). Determination of equid herpesvirus 1-specific, CD8+, cytotoxic T lymphocyte precursor frequencies in ponies. Vet Immunol Immunopathol 70, 43–54.[CrossRef][Medline]
Paillot, R., Daly, J. M., Juillard, V., Minke, J. M., Hannant, D. & Kydd, J. H. (2005). Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1. Vaccine 23, 4541–4551.[CrossRef][Medline]
Paillot, R., Ellis, S. A., Daly, J. M., Audonnet, J. C., Minke, J. M., Davis-Poynter, N., Hannant, D. & Kydd, J. H. (2006). Characterisation of CTL and IFN
synthesis in ponies following vaccination with a NYVAC-based construct coding for EHV-1 immediate early gene, followed by challenge infection. Vaccine 24, 1490–1500.[CrossRef][Medline]
Patel, J. R. & Heldens, J. (2005). Equine herpesviruses 1 (EHV-1) and 4 (EHV-4) – epidemiology, disease and immunoprophylaxis: a brief review. Vet J 170, 14–23.[CrossRef][Medline]
Piccini, A., Perkus, M. E. & Paoletti, E. (1987). Vaccinia virus as an expression vector. Methods Enzymol 153, 545–563.[Medline]
Rappocciolo, G., Birch, J. & Ellis, S. A. (2003). Down-regulation of MHC class I expression by equine herpesvirus-1. J Gen Virol 84, 293–300.
Sinclair, R., Cook, R. F. & Mumford, J. A. (1989). The characterization of neutralizing and non-neutralizing monoclonal antibodies against equid herpesvirus type 1. J Gen Virol 70, 455–459.
Slater, J. D., Borchers, K., Thackray, A. M. & Field, H. J. (1994). The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse. J Gen Virol 75, 2007–2016.
Soboll, G., Whalley, J. M., Koen, M. T., Allen, G. P., Fraser, D. G., Macklin, M. D., Swain, W. F. & Lunn, D. P. (2003). Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes. J Gen Virol 84, 2625–2634.
Tallmadge, R. L., Lear, T. & Antczak, D. F. (2005). Genomic characterization of MHC class I genes of the horse. Immunogenetics 57, 763–774.[CrossRef][Medline]
Tartaglia, J., Perkus, M. E., Taylor, J. & 9 other authors (1992). NYVAC: a highly attenuated strain of vaccinia virus. Virology 188, 217–232.[CrossRef][Medline]
Telford, E. A., Watson, M. S., McBride, K. & Davison, A. J. (1992). The DNA sequence of equine herpesvirus-1. Virology 189, 304–316.[CrossRef][Medline]
Thomson, G. R., Mumford, J. A., Campbell, J., Griffiths, L. & Clapham, P. (1976). Serological detection of equid herpesvirus 1 infections of the respiratory tract. Equine Vet J 8, 58–65.[Medline]
Welch, H. M., Bridges, C. G., Lyon, A. M., Griffiths, L. & Edington, N. (1992). Latent equid herpesviruses 1 and 4: detection and distinction using the polymerase chain reaction and co-cultivation from lymphoid tissues. J Gen Virol 73, 261–268.
Zhang, Y., Smith, P. M., Tarbet, E. B., Osterrieder, N., Jennings, S. R. & O'Callaghan, D. J. (1998). Protective immunity against equine herpesvirus type-1 (EHV-1) infection in mice induced by recombinant EHV-1 gD. Virus Res 56, 11–24.[CrossRef][Medline]
Received 27 March 2006;
accepted 2 May 2006.
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) or singly transfected with a plasmid expressing either MHC class I gene B2 (
) or EHV-1 gene 64 (
). Mean CTL activity±standard deviation (vertical bars) of at least three replicate cultures. Effector-to-target ratio is shown on the x axis. (a) Pony 6C31 (B2+) and (b) thoroughbred AM (B2+) lysed the double, but not single transfectants. (c) Pony 302A (B2–) failed to lyse any transfectants.
