Immediate-early protein of equid herpesvirus type 1 as a target for cytotoxic T-lymphocytes in the Thoroughbred horse.

Cytotoxic T-lymphocytes (CTLs) are associated with protective immunity against disease caused by equid herpesvirus type 1 (EHV-1). However, the EHV-1 target proteins for CTLs are poorly defined. This limits the development of vaccine candidates designed to stimulate strong CTL immunity. Here, classical CTL assays using lymphocytes from horses of three defined MHC class I types that experienced natural infection with EHV-1 and a modified vaccinia virus construct containing an EHV-1 gene encoding the immediate-early (IE) protein are reported. Horses homozygous for the equine leukocyte antigen (ELA)-A2 haplotype, but not the ELA-A5 haplotype, produced MHC-restricted CTL responses against the IE protein. Previously, horses homozygous for the ELA-A3 haplotype also mounted CTL responses against the IE protein. Both haplotypes are common in major horse breeds, including the Thoroughbred. Thus, the IE protein is an attractive candidate molecule for future studies of T-cell immunity to EHV-1 in the horse.

Vaccination of foals with a modified live, equid herpesvirus-1 gM deletion mutant (RacHΔgM) confers partial protection against infection.

Equid herpesvirus-1 (EHV-1) causes respiratory and neurological disease and late gestation abortion in pregnant mares. Current vaccines contain either inactivated or live EHV-1, but fail to provide complete clinical or virological protection, namely prevention of nasopharyngeal shedding and cell-associated viraemia. Thus, the development of novel products, such as modified live virus (MLV) vaccines which stimulate virus-specific, humoral and cell mediated immune responses more effectively remains a priority. Two groups of weaned foals (n = 6 each group) were used in a longitudinal, prospective, experimental study to evaluate immune responses elicited by two vaccinations with a glycoprotein M (gM) deletion mutant of EHV-1 (RacHdeltagM). Following two concurrent intranasal and intramuscular inoculations six weeks apart, vaccinated (8.4 ±â€¯0.2 months old) and control foals (6.2 ±â€¯0.4 months) were challenge infected intranasally with EHV-1 Ab4/8 four weeks after the second vaccination and clinical signs and virological replication measured. Vaccination caused no adverse events, but did stimulate significantly higher complement fixing and virus neutralizing antibodies in serum compared with control foals at either equivalent or pre-vaccination time points. Virus-specific nasopharyngeal antibody levels and cytotoxic T lymphocyte responses were not significantly different between the groups. Following challenge infection, these immune responses were associated with a reduction in clinical signs and virological replication in the vaccinated foals, including a reduction in duration and magnitude of pyrexia, nasopharyngeal shedding and cell-associated viraemia. We conclude that the RacHΔgM MLV primed EHV-1-specific humoral immune responses in weaned foals. However, complete virological protection by vaccination against EHV-1 requires further research.

A point mutation in a herpesvirus polymerase determines neuropathogenicity.

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.

Frequency and phenotype of EHV-1 specific, IFN-gamma synthesising lymphocytes in ponies: the effects of age, pregnancy and infection.

Equine herpesvirus-1 (EHV-1) infects horses, causing acute respiratory disease, neurological signs, and is also a leading cause of abortion. Protection from EHV-1 infection and disease depends on both humoral (virus neutralising antibody) and cellular (mainly cytotoxic T lymphocytes, CTL) immune responses. CTL activity after EHV-1 infection has been extensively investigated and is closely associated with an alternative measure of cell mediated immunity (CMI), interferon-gamma (IFN-gamma) synthesis. This study investigates EHV-1-specific IFN-gamma synthesising cells in potentially immunocompromised horses; foals, pregnant mares and aged animals, after field or experimental infection with EHV-1. In foals and pregnant mares, the kinetics after experimental infection were similar and the phenotype of IFN-gamma+ synthesising cells after EHV-1 stimulation was mainly CD8alpha+. In contrast, in samples collected from primed healthy ponies exposed to EHV-1 several months previously or in old ponies (28 years old), the majority of EHV-1-specific IFN-gamma+ lymphocytes expressed a CD5+, CD8alpha- phenotype. This study highlights the complexity of the relationship between EHV-1, a common pathogen in horses, and the virus-specific cellular immune response as measured using IFN-gamma synthesis.

Further characterization of cross-reactive anti-human leukocyte mAbs by use of equine leukocyte cell lines EqT8888 and eCAS.

The equine leukocyte cell lines, namely eCAS (bone marrow derived) and EqT8888 (lymphoma derived) were used for further analysis, using 40 commercially available mAbs that showed reactivity with equine PBMC. Most mAbs that were detected in these previous studies to react with PBMC, however, did not react with either of these cell lines. Fifteen mAbs were positive on at least one of the cell lines and indicate opportunities to set up further tests using these cells. Notably, two mAbs directed against human CD34 were detected to react with eCAS.

Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes.

Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.

The equine immune response to equine herpesvirus-1: the virus and its vaccines.

Equine herpesvirus-1 (EHV-1) is an alphaherpesvirus which infects horses, causing respiratory and neurological disease and abortion in pregnant mares. Latency is established in trigeminal ganglia and lymphocytes. Immunity to EHV-1 lasts between 3 and 6 months. Current vaccines, many of which contain inactivated virus, have reduced the incidence of abortion storms in pregnant mares but individual animals, which may be of high commercial value, remain susceptible to infection. The development of effective vaccines which stimulate both humoral and cellular immune responses remains a priority. Utilising data generated following experimental and field infections of the target species, this review describes the immunopathogenesis of EHV-1 and the interaction between the horse's immune system and this virus, both in vivo and in vitro, and identifies immune responses, highlighting those which have been associated with protective immunity. It then goes on to recount a brief history of vaccination, outlines factors likely to influence the outcome of vaccine administration and describes the immune response stimulated by a selection of commercial and experimental vaccines. Finally, based on the available data, a rational strategy designed to stimulate protective immune responses by vaccination is outlined.

A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.