Identification of the immunodominant H-2K(k)-restricted cytotoxic T-cell epitope in the Borna disease virus nucleoprotein

Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8(+) T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2K(k)-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensitivity to lysis by CTLs isolated from brains of BDV-infected MRL mice with acute neurological disease. Vaccinia virus-mediated expression of a p40 variant with mutations in the two K(k)-specific anchor residues of the TELEISSI peptide (p40(E130K,I136T)) did not sensitize L929 target cells for lysis by BDV-specific CTLs, whereas expression of wild-type p40 did. Furthermore, unlike vaccination with wild-type p40, vaccination of persistently infected symptomless B10.BR mice with p40(E130K,I136T) did not result in central nervous system inflammation and neurological disease. These results demonstrate that TELEISSI is the immunodominant CTL epitope of BDV p40 in H-2k mice.     

CD8 T cells require gamma interferon to clear borna disease virus from the brain and prevent immune system-mediated neuronal damage

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.     

Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease

The nucleoprotein (N) of Borna disease virus (BDV) is the major target of the disease-inducing antiviral CD8 T-cell response in the central nervous system of mice. We established two transgenic mouse lines which express BDV-N in either neurons (Neuro-N) or astrocytes (Astro-N). Despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. When Neuro-N mice were infected as adults, replication of BDV was severely impaired and was restricted to brain areas with a low density of transgene-expressing cells. Notably, the virus failed to replicate in the transgene-expressing granular and pyramidal neurons of the hippocampus (which are usually the preferred host cells of BDV). When Neuro-N mice were infected within the first 5 days of life, replication of BDV was not suppressed in most neurons, presumably because the onset of transgene expression in the brain occurred after these cells became infected with BDV. Astro-N mice remained susceptible to BDV infection, but they were resistant to BDV-induced neurological disorder. Unlike their nontransgenic littermates, Neuro-N mice with persistent BDV infection did not develop neurological disease after immunization with a vaccinia virus vector expressing BDV-N. In contrast to the situation in wild-type mice, this treatment also failed to induce N-specific CD8 T cells in the spleens of both transgenic mouse lines. Thus, while resistance to BDV infection in N-expressing neurons appeared to result from untimely expression of a viral nucleocapsid component, the resistance to BDV-induced neuropathology probably resulted from immunological tolerance.     

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.     

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.     

Borna Disease Virus-Induced Neurological Disorder in Mice: Infection of Neonates Results in Immunopathology

Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that persistently infects warm-blooded animals. In horses and other natural animal hosts, infections with BDV cause meningoencephalitis and behavioral disturbances. Experimental infection of adult mice takes a nonsymptomatic course, an observation previously believed to indicate that this animal species is not suitable for pathogenesis studies. We now demonstrate that BDV frequently induces severe neurological disease in infected newborn mice. Signs of neurological disease were first observed 4 to 6 weeks after intracerebral infection. They included a characteristic nonphysiological position of the hind limbs at an early stage of the disease and paraparesis at a later stage. Histological examination revealed large numbers of perivascular and meningeal inflammatory cells in brains of diseased mice and, unexpectedly, no increase in immunoreactivity to glial fibrillar acidic protein. The incidence and severity of BDV-induced disease varied dramatically among mouse strains. While only 13% of the infected C57BL/6 mice showed disease symptoms, which were mostly transient, more than 80% of the infected MRL mice developed severe neurological disorder. In spite of these differences in susceptibility to disease, BDV replicated to comparable levels in the brains of mice of the various strains used. Intracerebral infections of newborn β2-microglobulin-deficient C57BL/6 and MRL mice, which both lack CD8⁺ T cells, did not result in meningoencephalitis or neurological disease, indicating that the BDV-induced neurological disorder in mice is a cytotoxic T-cell-mediated immunopathological process. With this new animal model it should now be possible to characterize the disease-inducing immune response to BDV in more detail.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

CD8(+) T lymphocytes mediate Borna disease virus-induced immunopathology independently of perforin

Perforin-mediated lysis of target cells is the major antiviral effector mechanism of CD8(+) T lymphocytes. We have analyzed the role of perforin in a mouse model for CD8(+) T-cell-mediated central nervous system (CNS) immunopathology induced by Borna disease virus. When a defective perforin gene was introduced into the genetic background of the Borna disease-susceptible mouse strain MRL, the resulting perforin-deficient mice developed strong neurological disease in response to infection indistinguishable from that of their perforin-expressing littermates. The onset of disease was slightly delayed. Brains of diseased perforin-deficient mice showed similar amounts and a similar distribution of CD8(+) T cells as wild-type animals. Perforin deficiency had no impact on the kinetics of viral spread through the CNS. Unlike brain lymphocytes from diseased wild-type mice, lymphocytes from perforin-deficient MRL mice showed no in vitro cytolytic activity towards target cells expressing the nucleoprotein of Borna disease virus. Taken together, these results demonstrate that CD8(+) T cells mediate Borna disease independent of perforin. They further suggest that the pathogenic potential of CNS-infiltrating CD8(+) T cells does not primarily reside in their lytic activity but rather in other functions.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Increased epitope-specific CD8+ T cells prevent murine coronavirus spread to the spinal cord and subsequent demyelination

CD8+ T cells are important for clearance of neurotropic mouse hepatitis virus (MHV) strain A59, although their possible role in A59-induced demyelination is not well understood. We developed an adoptive-transfer model to more clearly elucidate the role of virus-specific CD8+ T cells during the acute and chronic phases of infection with A59 that is described as follows. C57BL/6 mice were infected with a recombinant A59 virus expressing the gp33 epitope, an H-2Db-restricted CD8+ T-cell epitope encoded in the glycoprotein of lymphocytic choriomeningitis virus, as a fusion with the enhanced green fluorescent protein (RA59-gfp/gp33). P14 splenocytes (transgenic for a T-cell receptor specific for the gp33 epitope) were transferred at different times pre- and postinfection (p.i.). Adoptive transfer of P14 splenocytes 1 day prior to infection with RA59-gfp/gp33, but not control virus lacking the gp33 epitope, RA59-gfp, reduced weight loss and viral replication and spread in the brain and to the spinal cord. Furthermore, demyelination was significantly reduced compared to that in nonrecipients. However, when P14 cells were transferred on day 3 or 5 p.i., no difference in acute or chronic disease was observed compared to that in nonrecipients. Protection in mice receiving P14 splenocytes prior to infection correlated with a robust gp33-specific immune response that was not observed in mice receiving the later transfers. Thus, an early robust CD8+ T-cell response was necessary to reduce virus replication and spread, specifically to the spinal cord, which protected against demyelination in the chronic phase of the disease.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1.

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.     

DM peptide-editing function leads to immunodominance in CD4 T cell responses in vivo

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158-173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM-/-) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33-48 and 261-276). DM-/- B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.     

CD4+ T-cell-epitope escape mutant virus selected in vivo.

Mutations in viral genomes that affect T-cell-receptor recognition by CD8+ cytotoxic T lymphocytes have been shown to allow viral evasion from immune surveillance during persistent viral infections. Although CD4+ T-helper cells are crucially involved in the maintenance of effective cytotoxic T-lymphocyte and neutralizing-antibody responses, their role in viral clearance and therefore in imposing similar selective pressures on the virus is unclear. We show here that transgenic virus-specific CD4+ Tcells, transferred into mice persistently infected with lymphocytic choriomeningitis virus, select for T-helper epitope mutant viruses that are not recognized. Together with the observed antigenic variation of the same T-helper epitope during polyclonal CD4+ T-cell responses in infected pore-forming protein-deficient C57BL/6 mice, this finding indicates that viral escape from CD4+ T lymphocytes is a possible mechanism of virus persistence.     

Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.     

Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and enhances antiviral protection

In herpesvirus infections, the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection, we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist, we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly, persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice, indicating enhanced rather than diminished effector functions. Consistent with this, virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed, such as changes in Bcl-2 levels, interleukin-2 production, and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as "classical" memory CD8(+) T cells.     

Borna disease virus multiplication in mouse organotypic slice cultures is site-specifically inhibited by gamma interferon but not by interleukin-12

Borna disease virus (BDV) induces a nonpurulent CD4- and CD8-T-cell-dependent meningoencephalitis in susceptible animals. Upon intracerebral infection, BDV replicates in the mouse central nervous system (CNS), but only a few mouse strains develop neurological disorder. The antiviral T cells appear to suppress BDV replication by a noncytolytic mechanism. Since BDV does not replicate in standard mouse cell cultures, the putative role of gamma interferon (IFN-gamma) in virus control could not be tested experimentally. Here, we report that mouse organotypic slice cultures can be used to elucidate the complex interactions of BDV, the CNS, and the immune system. We show that BDV replicated in various cell types of mouse cerebellar slice cultures in vitro. In infected slice cultures, a moderate upregulation of the chemokine genes CCL5 and CXCL10 was observed, while expression of various neural genes as well as other chemokine and cytokine genes was not altered. IFN-gamma inhibited the multiplication of BDV in cerebellar and hippocampal slice cultures in a dose-dependent manner. However, while complete suppression of BDV was observed in cerebellar slice cultures, inhibition was incomplete in hippocampal slice cultures. Kinetic studies indicated that IFN-gamma protects noninfected cells from infection rather than clearing the virus from infected cells. These results demonstrate that BDV can replicate in cultured neural cells of the mouse if organ integrity is well preserved. They further show that IFN-gamma is a powerful inhibitor of BDV in the absence of blood-borne leukocytes in mouse cerebellar slice cultures.     

Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants

Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.