Role of CTL mutants in demyelination induced by mouse hepatitis virus, strain JHM.

Mouse hepatitis virus, strain JHM (MHV-JHM) is a well described cause of demyelination. C57B1/6 (B6) mice infected at the suckling stage in the presence of protective antibodies remain asymptomatic initially but later develop clinical disease (hindlimb paralysis). Infectious virus can be isolated from these mice. Recently, two MHV-specific target epitopes for cytotoxic CD8 T cells have been identified in B6 mice. Our results show that in all mice with hindlimb paralysis, mutations can be detected in the RNA encoding the immunodominant of the two epitopes. These mutations result in a loss of recognition by MHV-specific cytotoxic T cells. These changes are not detected, for the most part, in mice that remain asymptomatic nor in mice with acute encephalitis. These results suggest that the development of CTL escape mutants is necessary for hindlimb paralysis to develop in this model.

Immune response to the immunodominant epitope of mouse hepatitis virus is polyclonal, but functionally monospecific in C57Bl/6 mice.

Mutations in an immunodominant CD8 CTL epitope (S-510-518) are selected in mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus. These mutations abrogate recognition by T cells harvested from the infected CNS in direct ex vivo cytotoxicity assays. Previous reports have suggested that, in general, an oligoclonal, monospecific T cell response contributes to the selection of CTL escape mutants. Herein, we show that, in MHV-JHM-infected mice, the CD8 T cell response after intraperitoneal infection is polyclonal and diverse. This diverse response was shown to include both polyclonal and oligoclonal components. The polyclonal data were shown to fit a logarithmic distribution. With regard to specificity, we used a panel of peptide analogues of epitope S-510-518 and spleen-derived CD8 T cell lines to determine why only a subset of possible mutations was selected in persistently infected mice. At a given position in the epitope, the mutations identified in in vivo isolates were among those that resulted in the greatest loss of recognition. However, not all such mutations were selected, suggesting that additional factors must contribute to selection in vivo. By extrapolation of these results to the persistently infected CNS, they suggest that the selection of CTL escape mutants requires the presence of a monospecific T cell response but also show that this response need not be oligoclonal.

Cytotoxic T-cell-resistant variants arise at early times after infection in C57BL/6 but not in SCID mice infected with a neurotropic coronavirus.

Under certain conditions, C57BL/6 mice persistently infected with mouse hepatitis virus strain JHM (MHV-JHM) develop clinical disease and histological evidence of demyelination several weeks after inoculation with virus. In a previous report, we showed that mutations in the RNA encoding an immunodominant CD8 T-cell epitope within the surface glycoprotein (epitope S-510-518) were present in all persistently infected animals and that these mutations abrogated recognition by virus-specific cytotoxic T cells (CTLs) in direct ex vivo cytotoxicity assays. To obtain further evidence that these mutations were necessary for the development of clinical disease, the temporal course of their appearance was determined. Mutations in the epitope were identified by 10 to 12 days after inoculation, and in some mice, virus containing mutated epitope was the dominant species detected by 15 days. In addition, most mice that remain asymptomatic at 80 days after inoculation, a time after which clinical disease almost never develops, were infected with only wild-type virus. Finally, analysis of virus isolated from mice with severe combined immunodeficiency (SCID) revealed the presence only of wild-type epitope S-510-518. These results, by showing that mutations are not selected in SCID mice and occur at early times after inoculation in C57BL/6 mice, support the view that they result from immune pressure and contribute to virus persistence and demyelination in mice infected persistently with MHV-JHM.

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.

High-magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice.

The neurotropic JHM strain of mouse hepatitis virus (MHV) causes acute encephalitis and chronic demyelinating encephalomyelitis in rodents. Previous results indicated that CD8 T cells infiltrating the central nervous system (CNS) were largely antigen specific in both diseases. Herein we show that by 7 days postinoculation, nearly 30% of the CD4 T cells in the acutely infected CNS were MHV specific by using intracellular gamma interferon (IFN-gamma) staining assays. In mice with chronic demyelination, 10 to 15% of the CD4 T cells secreted IFN-gamma in response to MHV-specific peptides. Thus, these results show that infection of the CNS is characterized by a large influx of CD4 T cells specific for MHV and that these cells remain functional, as measured by cytokine secretion, in mice with chronic demyelination.

Coronavirus-induced demyelination occurs in the absence of inducible nitric oxide synthase.

Demyelination induced by mouse hepatitis virus (MHV), strain JHM, is in large part immune mediated, but little is known about the mechanisms involved in this process. Previous results suggest that inducible nitric oxide synthase (NOS2) contributes transiently to MHV-induced demyelination. Herein, we show that equivalent amounts of demyelination were evident at day 12 after MHV infection in mice genetically deficient in NOS2 (NOS2(-/-)) and in C57BL/6 mice. Furthermore, using an established adoptive transfer model and pharmacological inhibitors of NOS2 function, we could demonstrate no effect on MHV-induced demyelination. These results indicate that NOS2 function is not required for demyelination in mice infected with MHV.

Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease.

C57BI/6 mice infected with mouse hepatitis virus, strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis. Infectious virus can be isolated only from symptomatic mice. In C57BI/6 mice, two CD8+ T cell epitopes within the MHV-JHM surface glycoprotein were previously identified. Here, we show that mutations in the RNA encoding the immunodominant of the epitopes are present in nearly all virus samples isolated from these mice. Mutations are not present in sequences flanking this epitope or in other CD8+ or CD4+ T cell epitopes. Furthermore, analysis of five peptides corresponding to variant epitopes in direct ex vivo cytotoxicity assays showed that each mutation caused a loss of epitope recognition. These results suggest that escape from CD8+ T cell recognition is necessary for enhanced virus replication and development of clinical disease in these MHV-JHM-infected mice.

CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination.

A chronic demyelinating disease results from murine infection with the neurotropic strain JHM of mouse hepatitis virus (MHV-JHM). Demyelination is largely immune mediated. In this study, the individual roles of CD4 and CD8 T cells in MHV-induced demyelination were investigated using recombination-activating gene 1-/- (RAG1-/-) mice infected with an attenuated strain of MHV-JHM. These animals develop demyelination only after adoptive transfer of splenocytes from mice previously immunized to MHV. In this study, we show that, following adoptive transfer, virus-specific CD4 and CD8 T cells rapidly infiltrate the CNS of MHV-JHM-infected RAG1-/- mice. Adoptive transfer of CD4 T cell-enriched donors resulted in more severe clinical disease accompanied by less demyelination than was detected in the recipients of undepleted cells. Macrophage infiltration into the gray matter of CD4 T cell-enriched recipients was greater than that observed in mice receiving undepleted splenocytes. In contrast, CD8 T cell-enriched recipients developed delayed disease with extensive demyelination of the spinal cord. MHV-JHM-infected RAG1-/- mice receiving donors depleted of both CD4 and CD8 T cells did not develop demyelination. These results demonstrate that the development of demyelination following MHV infection may be initiated by either CD4 or CD8 T cells. Furthermore, they show that CD4 T cells contribute more prominently than CD8 T cells to the severity of clinical disease, and that this correlates with increased macrophage infiltration into the gray matter.

Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus.

Mice infected with mouse hepatitis virus (MHV) strain JHM develop primary demyelination. Herein we show that axonal damage occurred in areas of demyelination and also in adjacent areas devoid of myelin damage. Immunodeficient MHV-infected RAG1-/- mice (mice defective in recombinase activating gene 1 expression) do not develop demyelination unless they receive splenocytes from a mouse previously immunized against MHV (G. F. Wu, A. Dandekar, L. Pewe, and S. Perlman, J. Immunol. 165:2278-2286, 2000). In the present study, we show that adoptive transfer of T cells was also required for the majority of the axonal injury observed in these animals. Both demyelination and axonal damage were apparent by 7 days posttransfer. Recent data suggest that axonal injury is a major factor in the long-term disability observed in patients with multiple sclerosis. Our data demonstrate that immune system-mediated damage to axons is also a common feature in mice with MHV-induced demyelination. Remarkably, there appeared to be a minimal, if any, interval of time between the appearance of demyelination and that of axonal injury.

Selection of CTL escape mutants in mice infected with a neurotropic coronavirus: quantitative estimate of TCR diversity in the infected central nervous system.

Variant viruses mutated in the immunodominant cytotoxic T cell epitope surface (S) glycoprotein S-510-518 are selected in mice chronically infected with mouse hepatitis virus, strain JHM. We determined whether this selection occurred in the presence of an oligoclonal or polyclonal T cell response using soluble MHC/peptide tetramers in direct ex vivo analyses of CNS-derived lymphocytes. A total of 42% (range, 29-60%) of CD8 T cells in the CNS of mice with acute encephalitis recognized epitope S-510-518. A total of 34% (range, 18-62%) of cells from mice with hind limb paralysis (and chronic demyelination) were also epitope specific, even though only virus expressing mutated epitope is detected in these animals. Sequence analysis of the beta-chain CDR3 of 487 tetramer S-510-518-positive cDNA clones from nine mice showed that a majority of clonotypes were identified in more than one mouse. From these analyses, we estimated that 300-500 different CD8 T cell clonotypes responsive to epitope S-510-518 were present in each acutely infected brain, while 100-900 were present in the CNS of each mouse with chronic disease. In conclusion, a polyclonal CD8 T cell response to an epitope does not preclude the selection of T cell escape mutants, and epitope-specific T cells are still present at high levels even after RNA-encoding wild-type sequence is no longer detectable.

Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis.

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.