Characterization of the Cytolytic T-Lymphocyte Response to a Candidate Vaccine Strain of Equine Herpesvirus 1 in CBA Mice

The cytolytic T-lymphocyte (CTL) response to respiratory infection with equine herpesvirus 1 (EHV-1) in CBA (H-2^k) mice was investigated. Intranasal (i.n.) inoculation of mice with the attenuated EHV-1 strain KyA resulted in the generation of a primary virus-specific CTL response in the draining mediastinal lymph nodes 5 days following infection. EHV-1-specific CTL could be restimulated from the spleen up to 26 weeks after the resolution of infection, indicating that a long-lived memory CTL population was generated. Depletion of CD8⁺ T cells by treatment with antibody and complement prior to assay eliminated CTL activity from both primary and memory populations, indicating that cytolytic activity in this model was mediated by class I major histocompatibility complex-restricted, CD8⁺ T cells. A single i.n. inoculation with KyA induced protective immunity against infection with the pathogenic EHV-1 strain, RacL11. The adoptive transfer of splenocytes from KyA-immune donors into sublethally irradiated recipients resulted in a greater than 250-fold reduction in RacL11 in the lung. The elimination of both CD4⁺ and CD8⁺ T cells from the transferred cells abrogated clearance of RacL11, while the selective depletion of either subpopulation alone had little effect. These results suggested that both lymphocyte subpopulations contribute to viral clearance, with either subpopulation alone being sufficient.Equine herpesvirus 1 (EHV-1) is a prevalent respiratory pathogen of horses worldwide (8, 17, 38). Infection of horses with EHV-1 results in fever, respiratory distress, abortagenic disease, and severe neurological sequelae (3, 13, 27, 35, 36). The highly contagious respiratory transmission of EHV-1 has resulted in disastrous outbreaks of disease in domestic horse populations and has had a significant economic impact on the equine industry. EHV-1 infection of the horse results in the generation of a short-lived humoral response but does not confer long-term protection, as disease often occurs following natural infection (10, 22). Although both live and inactivated vaccines are currently available for EHV-1, only relatively short-lived protection has been observed (11, 12, 24). Furthermore, it is not clear which immune functions are responsible for conferring the short-lived protection following vaccination. In addition to specific antibody responses, peripheral blood leukocytes from vaccinated horses produce gamma interferon in culture (19). EHV-1-specific, CD8⁺ class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) have been identified in peripheral blood mononuclear cells, reaching maximal levels 2 to 3 weeks postinfection (p.i.) (4, 20). However, the effectiveness of the current vaccines in stimulating EHV-1-specific CTL and their role in protective immunity in vivo are currently not known.Horses inoculated with the attenuated EHV-1 strain Kentucky A (KyA) exhibited a reduction in clinical signs following challenge with a pathogenic EHV-1 strain (33, 34). Although the attenuated strain induced a protective response, in terms of a reduced duration of viral shedding and viremia, the ability of this strain to induce an EHV-1-specific antibody response was weaker than that of the virulent strain. This finding suggested that immune functions other than the generation of specific antibodies might be critical in the resolution of infection. To generate a more effective EHV-1 vaccine, a better understanding of the precise immune functions associated with protection and resolution from EHV-1 infection is essential.A murine model of respiratory EHV-1 infection which closely mimicked EHV-1 infection in the natural host was established in various strains of mice (5). Common features included replication in the respiratory mucosae, the development of pneumonitis, cell-associated viremia, and abortion (5, 6). Specific immune responses are important for modulating infection. In the mouse, the passive transfer of hyperimmune polyclonal rabbit EHV-1-specific antibodies into infected mice significantly reduced the viremia following challenge with live EHV-1 (5). Subsequent studies demonstrated that various EHV-1 glycoproteins, including gB, gC, gD and gH, were capable of inducing the generation of neutralizing antibodies (9, 23, 40, 49, 52, 56). Furthermore, inoculation of mice with gB, gC, and/or gD elicited a protective response against subsequent challenge with pathogenic EHV-1 (39, 49, 50, 52, 56). However, each of these EHV-1 gene products is capable of eliciting both B- and T-cell responses; thus, the role of distinct immune functions conferring protection is not clearly defined.In the most extensively studied BALB/c mouse model of EHV-1 infection, adoptive transfer experiments demonstrated that immune spleen cells isolated from mice primed with live, but not heat-killed, EHV-1 reduced viral levels in both the lungs and nasal turbinates of infected recipient mice (5). Although the specific cell population responsible for protection was not determined, the data suggested that cell-mediated immune functions may be critical in the resolution of EHV-1 infection. The adoptive transfer of defined T-cell subpopulations demonstrated that both CD4⁺ and CD8⁺ T cells play a role in controlling EHV-1 respiratory infection. The CD8⁺ T-cell subpopulation appeared to play a more dominant role, although the functions by which protection was mediated were not defined (7). If the immune response was elicited by immunization with recombinant baculovirus-derived EHV-1 glycoproteins, the response was altered so that CD4⁺ T cells were predominantly associated with protection (52). Thus, either T-cell subpopulation is likely to play an important role in the optimal response to infection.While adoptive transfer studies have implicated an important role for CD8⁺ T cells in the control of EHV-1 infection in the lungs of BALB/c mice (7), there has been no direct assessment of CD8⁺ T-cell effector functions in this model. This is due predominantly to the lack of suitable class I MHC-compatible, H-2^d-expressing murine cells that are susceptible to infection with EHV-1. However, the attenuated KyA strain of EHV-1 has been propagated in our laboratory in suspension cultures of murine L-M fibroblasts that express the H-2^k haplotype. Therefore, an alternative mouse model, using mice expressing the H-2^k haplotype, was adopted principally to assess the activation of EHV-1-specific CTL responses. Initial studies by Awan et al. (5) demonstrated that CBA (H-2^k) mice were susceptible to infection with EHV-1 strain Ab4. In the present study, CBA mice were susceptible to respiratory tract infection by both the nonpathogenic KyA and the pathogenic RacL11 strains of EHV-1. Furthermore, infection of CBA mice with the attenuated KyA strain generated a vigorous CD8⁺, class I MHC-restricted, EHV-1-specific primary CTL response in the draining mediastinal lymph nodes (MLN) and a long-term memory CTL response in the spleen. These studies provide the basis for a model system to analyze the potential importance of class I MHC-restricted CTL activity in controlling EHV-1 infection in vivo.