Protection against Lethal Encephalomyocarditis Virus Infection in the Absence of Serum-Neutralizing Antibodies

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAβ^−/−) mice, which are unable to mediate CD4⁺ T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAβ^−/− mice inoculated with 10⁴ PFU of attenuated Mengo virus (vMC₂₄) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8⁺ T lymphocytes prior to lethal EMCV challenge ablated protection in vMC₂₄-immunized RHAβ^−/− mice. The CD8⁺ T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8⁺ T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8⁺ T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC₂₄-immunized RHAβ^−/− mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC₂₄-immunized RHAβ^−/− mice survived when rechallenged 90 days later. The present study demonstrating CD8⁺ T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC₂₄-specific CD4⁺ T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.Picornaviruses are a family of positive-strand RNA viruses that are responsible for a variety of devastating human and animal diseases. The family is divided into six genera, enteroviruses, hepatoviruses, parechoviruses, rhinoviruses, aphthoviruses, and cardioviruses, that include such members as poliovirus, human rhinovirus, foot-and-mouth disease virus, and encephalomyocarditis virus (EMCV) (42). Mice are highly susceptible and considered the natural host for cardioviruses such as Mengo virus and EMCV, (7, 35), infections with which result in acute neurotropic disease producing rapid and lethal meningoencephalomyelitis (16, 47). The ability to protect mice against cardiovirus-induced disease by the elicitation or passive transfer of neutralizing antibodies is well documented (2, 13, 26, 41). Current dogma asserts that prophylaxis against picornavirus infection is afforded by serum-neutralizing antibodies (23, 25, 28). Existing picornavirus vaccines (23, 25), in addition to current strategies using recombinant-attenuated and protein-subunit vaccines (27, 32), are designed to elicit a protective neutralizing antibody response to capsid determinants. Indeed, serum-neutralizing titers are used to evaluate host immune status to a particular picornavirus pathogen.Mengo virus and EMCV are members of a single cardiovirus serotype and are indistinguishable by immune sera (42). The dramatic attenuation of Mengo virus by a truncation in the 5′-noncoding-region poly(C) tract preserves complete integrity of all virally encoded proteins (10), allowing in vivo exposure of structural and nonstructural proteins that may elicit an immune response. Normal immunocompetent mice immunized with an attenuated strain of Mengo virus (vMC₂₄) elicit high serum-neutralizing antibody titers and are protected from lethal EMCV challenge (9, 29). In addition to invoking a potent humoral response, vMC₂₄ is also capable of eliciting a cell-mediated immune (CMI) response (29) as an immunodominant CD8⁺ cytotoxic T-lymphocyte (CTL) epitope has been recently identified in the VP2 capsid protein in vMC₂₄-immunized C57BL/6 mice (11).Earlier investigations of CMI responses to cardioviruses in T-cell deficiency models vacillated between elucidating the immunopathologic role that these cells may contribute in disease and discerning the beneficial aspects that T cells may mediate in protection. T-cell subset depletion of BALB/c mice with anti-CD4 or anti-CD8 antibodies prior to EMCV infection ameliorated clinical disease and reduced the frequency of demyelination (44), suggesting a participatory role for T cells in pathology. Conversely, mice rendered CD4 deficient prior to infection with Theiler’s murine encephalomyelitis virus (TMEV), another murine cardiovirus, failed to produce neutralizing antibodies; consequently, they were unable to clear virus from the central nervous system (CNS) and died from overwhelming encephalitis (49). Similarly, infection of major histocompatibility complex (MHC) class I (β₂-microglobulin)-deficient (β₂m^−/−) mice with TMEV indicates a requisite role for CD8⁺ T cells in viral clearance and suggests that CD8⁺ T cells are not major mediators in demyelination or disease (13, 39).More recently, researchers have begun to unveil the beneficial role that CD8⁺ T cells may have in resolving infection and immune protection. An early and abundant TMEV-specific CD8⁺ T-cell response is critical in determining the balance between viral persistence or resolution of infection (6, 22, 30). Using vMC₂₄-immunized C57BL/6 mice, Escriou et al identified an immunodominant CD8⁺ CTL epitope (11) that is cross-reactive to the same VP2 epitope of TMEV (5), although VP2 epitope-immunized C57BL/6 mice were not fully protected from subsequent lethal Mengo virus challenge.The present study is a direct extension of our earlier observation (29) that vMC₂₄-specific CD4⁺ T cells are capable of adoptively transferring immune protection against lethal EMCV challenge in the absence of prophylactic levels of serum-neutralizing antibodies. Using MHC class II-deficient mice that lack CD4⁺ T cells and are incapable of T-cell-dependent humoral responses (15), we obtained evidence demonstrating CD8⁺ T cell-dependent protection against lethal EMCV infection in the absence of serum-neutralizing antibodies.