Multiple Pathways to Induction of Virus-Induced Autoimmune Demyelination: Lessons from Theiler's Virus Infection

doi:10.1006/jaut.2000.0489, available online at http://www.idealibrary.com on

Journal of Autoimmunity (2001) 16, 219–227

Multiple Pathways to Induction of Virus-Induced Autoimmune Demyelination: Lessons from Theiler’s Virus Infection Stephen D. Miller, Julie K. Olson and J. Ludovic Croxford Departments of Microbiology-Immunology and the Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611, USA Key words: autoimmunity, epitope spreading, molecular mimicry, Theiler’s murine encephalomyelitis virus Abbreviations: APL, altered peptide ligand; CFA, complete Freund’s adjuvant; CNS, central nervous system; MS: multiple sclerosis; MSCH, mouse spinal cord homogenate; PLP, proteolipid protein; R-EAE, relapsing experimental autoimmune encephalomyelitis; TMEV, Theiler’s murine encephalomyelitis virus

Infection of SJL mice with wild-type BeAn strain of Theiler’s murine encephalomyelitis virus (TMEV) leads to CD4 + T cell-mediated CNS demyelination characterized by the development of anti-myelin epitope autoimmune responses via epitope spreading during the chronic stage of disease. To exmine the feasibility of virus-encoded mimic epitopes to initiate CNS autoimmunity, we recently developed a molecular mimicry model of virus-induced demyelinating disease wherein a non-pathogenic variant strain of TMEV was engineered to encode a 30-mer peptide encompassing the immunodominant myelin proteolipid protein, PLP139–151, epitope. SJL mice infected intracerebrally with TMEV encoding either the native PLP139–151 determinant or various peptide mimics of the epitope develop an early onset demyelinating disease mediated by activated PLP139–151-specific Th1 cells. The autoimmune nature of this early-onset demyelinating disease is shown by the fact that induction of tolerance to the PLP139–151 peptide prevents clinical disease and associated PLP139–151-specific T cell responses without affecting T cell reactivity to virus epitopes. Most significantly, TMEV encoding a molecular mimic peptide derived from the Haemophilus influenzae bacteria, homologous at only six out of thirteen of the core amino acids, led to CNS disease. These studies provide conclusive evidence that virus-induced myelinspecific autoreactive T cells can be induced by molecular mimicry and provide a useful model to study the disease inducing ability of viruses encoding human-disease-related mimicry peptides. © 2001 Academic Press

Introduction

epitopes shared or cross-reactive with self antigens, i.e. molecular mimicry [11, 12]. There is little experimental evidence to support a role for infectious agent encoded superantigens for initiating T cell-mediated autoimmunity, but there is growing evidence to support possible roles for either molecular mimicry or epitope spreading in autoimmune disease initiation. A schematic representation of the immunologic events occurring in these two phenomena is depicted in Figure 1. Our laboratory has been studying the pathogenesis of the immune-mediated CNS demyelinating disease which ensues following infection with Theiler’s murine encephalomyelitis virus (TMEV). We will review the data showing that infection of susceptible SJL mice with wild-type TMEV leads to initiation of myelin epitope-specific autoimmunity via epitope spreading. The characteristics of a new model of virus-induced molecular mimicry initiated following infection of SJL mice with a non-pathogenic TMEV variant engineered to encode an immunodominant encephalitogenic myelin peptide (PLP139–151) or various peptide mimics of this epitope will also be discussed.

The mechanism(s) underlying the initiation and progression of multiple sclerosis and other autoimmune diseases are not well understood, but epidemiologic studies have provided strong suggestive evidence for a role for virus infection(s) in the development and/or exacerbations of MS [1, 2]. Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by the presence of myelin epitope-specific CD4 + T cells in the central nervous system (CNS) [3, 4]. Postulated mechanisms of virus-induced autoimmunity include: stimulation of autoreactive T cells bearing particular V
receptors by virus-encoded superantigens [5]; de novo activation of autoreactive T cells by sequestered antigens released secondary to tissue destruction mediated directly by virus [6] or by virus-specific T cells, i.e. epitope spreading [7–10]; and activation of autoreactive T cells by pathogen-encoded Correspondence to: Dr Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. Fax: 312-503-1339. E-mail: [email protected] 219 0896–8411/01/030219+09 $35.00/0

© 2001 Academic Press

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Virus Peptide Acute Virus Infection

Figure 1. Schematic of molecular mimicry and epitope spreading models of virus-induced autoimmune disease. (A) In molecular mimicry, virus epitope-specific Th1 cells are activated and traffic across the blood-tissue barrier. Once in the tissue these virus-specific Th1 cells can recognize self peptides in a cross-reactive manner resulting in the release of cytokines and chemokines which chemo-attract and activate tissue resident and peripheral monocyte/macrophages which cause self tissue destruction and the resulting autoimmune disease. (B) In the epitope spreading model, virus epitope-specific Th1 cells are activated and traffic across the blood-tissue barrier where they recognize virus epitopes presented as a result of persistent tissue infection and initiate self tissue destruction. Self epitopes are released as a result of the initial tissue destruction and are processed and presented by both local and peripheral antigen-presenting cells resulting in the de novo activation of autoreactive Th1 cells which perpetuate the autoimmune disease.

Initiation of Myelin-Specific Autoimmunity via Epitope Spreading Following Infection with Wild-Type Theiler’s Virus TMEV is an endogenous mouse pathogen that establishes a life-long persistent CNS infection predominantly in CNS antigen-presenting cells such as microglia and macrophages. TMEV infection subsequently leads to the development of a progressive CD4 + T cell-mediated demyelinating disease which shares pathologic and immunologic similarities with the chronic-progressive form of human MS

[13, 14]. Clinical disease in SJL mice infected with wild-type TMEV normally develops 30–35 days post-infection and current evidence strongly indicates that initial CNS pathology is mediated by macrophage-mediated bystander destruction of myelin due to pro-inflammatory cytokines produced by TMEV-specific Th1 cells targeting CNS-persistent virus [15]. There are multiple lines of evidence indicating that neuroantigen-specific autoimmune T cells do not appear to play a role in initiation of TMEV disease. T cell responses to MBP and PLP epitopes are not detected prior to disease onset (30–35 days PI) [16, 17],

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Figure 2. Temporal pattern of clinical disease and virus- and myelin-epitope T cell responses in SJL mice infected with wild-type and PLP139–151-expressing viruses. (A) Following infection with wild-type TMEV, virus-specific Th1 cells are quickly activated and traffic to the CNS where they encounter virus epitopes presented by persistently infected macrophages/microglia leading to initial myelin destruction and onset of clinical disease beginning 30–35 days post-infection (see Figure 1). Initial myelin destruction then leads, via epitope spreading, to the de novo activation of myelin epitope-specific autoreactive Th1 cells which arise in an ordered progression. (B) Following infection with a PLP139–151 mimic epitope expressing TMEV, an early-onset (7–10 days post-infection) demyelinating disease is induced. Early Th1 responses are induced to both virus and to PLP139–151 both of which are encoded in the viral genome. The autoreactive PLP139–151 cells then traffic to the CNS where they cause the rapid onset of clinical disease. Responses to additional myelin epitopes are then induced via epitope spreading following the initial phase of myelin destruction.

while responses to TMEV epitopes are present by 7 days post-infection [18]. Peripheral tolerance to mouse spinal cord homogenate (MSCH—a heterogeneous mixture of multiple neuroantigens) does not affect the clinical onset of demyelination and the accompanying virus-specific T cell and antibody responses in TMEVinfected SJL/J mice [19]. However, this tolerogenic regimen is extremely effective in preventing clinical and histologic signs of MSCH-induced relapsing EAE (R-EAE) and the accompanying neuroantigen-specific DTH responses [19, 20]. In contrast, tolerance to intact TMEV virions coupled to syngeneic splenocytes, which specifically anergizes virus-specific Th1 responses [21, 22], results in a dramatic reduction in the incidence and severity of demyelinating lesions and clinical disease in SJL/J mice subsequently infected with TMEV [15]. Collectively, these results indicate that disease is initiated by virus-specific T cells targeting CNS persistent virus. Our more recent studies indicate that 3–4 weeks after the onset of clinical disease (i.e., approximately 50 days post-infection), when myelin damage reaches a critical threshold, CD4 + T cell responses to the immunodominant PLP139–151 epitope are induced and as disease progresses further, T cell responses to multiple myelin epitopes (e.g., PLP178–191, PLP56– 70, MOG92–106 and MBP84–104) arise in an ordered progression [7, 23]. Thus, autoimmune responses are only evident during the chronic stages of disease. A

schematic representation of the temporal appearance of virus- and myelin-specific T cell responses in relation to the clinical disease course in TMEV-infected SJL mice is depicted in Figure 2A. Following i.c. infection with the wild-type BeAn strain of TMEV, anti-myelin epitope responses arise via epitope spreading as indicated by the early appearance of responses to virus epitopes, but the late appearance of anti-myelin T cells. Additional data has shown a lack of cross-reactivity between T cells specific for TMEV epitopes and myelin proteins and peptides and vice versa [7]. In addition, the appearance of anti-myelin autoimmune responses is preceded by appearance of self epitopes on CNS APCs isolated from the spinal cords of affected mice indicating that virus-initiated myelin destruction leads to the processing and presentation of self epitopes in the CNS target organ which drive the epitope spreading process [23, 24]. Recent evidence shows that induction of peripheral tolerance to MP4 (a recombinant fusion protein comprising the 21.5 kD isoform of human MBP and a recombinant variant of human PLP) at 45 days postinfection ameliorates disease progression indicating that the anti-myelin autoimmune responses play an important functional pathologic role in chronic disease progression following infection with wild-type TMEV. Collectively, these results are highly significant since they provide compelling evidence that epitope spreading is an important alternative mechanism to

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molecular mimicry to explain the etiology of an organspecific autoimmune disease induced by a persistent CNS virus infection (Figure 1B).

Initiation of Myelin-Specific Autoimmunity by Virus-Induced Molecular Mimicry Molecular mimicry remains the major postulated mechanism by which infections may trigger autoimmune tissue damage [11, 25]. Evidence for a role for molecular mimicry in T cell-mediated autoimmune diseases stems mainly from experiments showing that mice expressing virus proteins as transgenes expressed in peripheral organs develop autoimmune disease after virus infection [26, 27] and from reports showing that immunization with viral peptides sharing limited sequence homology with self peptides can stimulate autoreactive T cells [25, 28, 29]. More recently, potential Borrelia burgdorferi mimic epitopes relevant to the pathogenesis of Lyme arthritis and neuroborreliosis have been identified using T cell clones [30, 31]. In addition, immunization of mice with a Chlamydia pneumoniae peptide was shown to induce heart disease via antigenic mimicry of a heart-muscle protein [32]. More direct evidence for infection-induced autoimmunity via molecular mimicry comes from recent evidence showing that infection of mice with herpes simplex virus type I (HSV-1) leads to the development of herpes stromal keratitis, an autoimmune eye disease [33]. HSV-1 coat protein-specific CD4 + T cells were shown to cross-react with an unidentified corneal antigen. We have recently developed a more controlled model to study the mechanisms of induction of autoimmunity via molecular mimicry. Our approach was to engineer a cDNA sequence encoding a 30 amino acid peptide (PLP130–159) encompassing the native encephalitogenic PLP139–151 epitope into a non-pathogenic variant of TMEV that had a Clal restriction site added and 23 amino acids deleted from the viral leader region (Clal-TMEV) (Figure 3). As a control, a virus expressing a 30-mer ovalbumin peptide (OVA317–346) was constructed. As tests for molecular mimicry, PLP139–151 epitope mimic viruses were constructed (Figure 1B) which had non-conservative amino acid substitutions at either position 147 (H147A) a secondary TCR recognition site sequence, or position 144 (W144A) the primary TCR recognition site [34, 35] and a virus encoding a PLP139–151 mimic peptide expressed by Haemophilus influenzae [36]. As indicated in Table 1, SJL mice infected intracerebrally with the wild-type BeAn strain of TMEV (WT BeAn) developed clinical signs of demyelination characterized by spastic paralysis which onset approximately 30–35 days post-infection while the non-pathogenic Clal parental virus containing the leader deletion did not induce clinical disease due to its inability to persist in the CNS. Interestingly, infection of SJL mice with a virus encoding 30mer

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cDNA encoding BeAn L P1 P2 Leader

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Figure 3. Schematic of construction of PLP139–151 mimic viruses. The cDNA encoding the BeAn strain of TMEV was modified to contain a Clal restriction site at bp1137 in the BeAn sequence and resulted in the deletion of 23 amino acids from the leader portion of the genome. This is the parental virus construct designated Clal-BeAn. PCR methods were used to insert Clal sites in the PLP DNA at both ends of a 30 amino acid piece, PLP130–159, which encompassed the encephalitogenic PLP139–151 epitope. The 30-amino acid piece containing PLP139–151 was then inserted into the Clal site in the Clal-BeAn virus cDNA. Mimic sequences of PLP139–151 were constructed by PCR mutagenesis of the PLP DNA to introduce alanine substitutions at position 144 (W144A) or position 147 (H147A) in the PLP DNA sequence prior to insertion into the Clal-BeAn viral genome at the Clal site. An additional molecular mimice peptide from Haemophilus influenzae (see Table 1 for the sequence) was also inserted in the Clal-BeAn virus cDNA. Ovalbumin peptide, OVA317–346, encompassing the OVA323–339 epitope, was inserted into the Clal site of Clal-BeAn, using the same methods as those for inserting PLP139–151, to produce a non-self epitope-containing virus (OVA323-BeAn). Viral RNA was produced from the cDNA through the T7 promoter and transfected into BHK-21 cells resulting in production of infectious virus by the cells.

encompassing a non-self OVA323–339 epitope led to similar appearing clinical disease, albeit with a slightly delayed onset (approx. 50 days post-infection) compared to the wild-type BeAn TMEV strain indicating that reintroduction of a non-viral 30mer sequence into the leader-deleted Clal virus can restore the ability of the virus to persist in vivo and cause clinical disease. Significantly mice infected with TMEV encoding PLP139–151 developed severe clinical signs of flaccid paralysis and histologic demyelination at very early times, i.e. 7–10 days post-infection as did mice infected with the H147A PLP139–151 altered peptide ligand (APL). Most significantly, TMEV encoding a PLP139–151 molecular mimic peptide derived from the Haemophilus influenzae bacterium, homologous at only six out of thirteen of the core amino acids including the primary TCR contact site at position 144 and the I-As contact residue at position 145, led to CNS disease. However, mice infected with the W144A PLP139–151 APL, containing a non-conservative amino acid substitution at the primary TCR contact residue, exhibited a delayed disease course similar to the virus containing the non-self OVA323–339 epitope.

Wildtype TMEV ClaI-TMEV OVA323-TMEV PLP139-TMEV H147A-TMEV H. influ.-TMEV W144A-TMEV

Mimic virus

None None ISQAVHAAHAEINEAGR HCLGKWLGHPDKF HCLGKWLGAPDKF EQLVKWLGLPAPI HCLGKALGHPDKF

Inserted mimic amino acid sequence

30–35 — 55–60 7–10 7–10 10–14 55–60

Day of onset

+ + + + + + +

d14 + + + + + + +

d60–d90

TMEV VP2 70–86

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d14 + + + + + + +

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PLP139–151

T cell response to virus and self peptides:

− − − − − − −

d14

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d60–d90

PLP178–191

Table 1. Capacity of TMEV expressing molecular mimics of PLP139-151 to induce early onset clinical disease and virus- and myelin-peptide-specific immune responses following infection of SJL mice

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The early-onset disease was also induced following infection with PLP139-TMEV by the intravenous and intraperitoneal routes indicating that the primary infection can be in sites distal from the CNS. The clinical results indicate that mice infected with a TMEV variant encoding the native PLP139–151 sequence or with a peptide mimic carrying a nonconservative amino acid substitution at the secondary TCR contact residue can induce an early-onset, EAE-like clinical disease. To determine whether the early onset disease was due to a ‘mimicry’ response against the immunodominant PLP139–151 epitope the temporal progression of antigen-specific T cell responses (T cell proliferation, IFN- production, and delayed-type hypersensitivity responses) to both virus and myelin epitopes was determined. As previously reported [7, 23], myelin-specific CD4 + Th1 responses in mice infected with wild-type TMEV are only first observed to the immunodominant PLP139–151 epitope beginning around 50 days post-infection, while responses to virus epitopes are demonstrable both early and late during infection. In contrast, mice infected with the native PLP139–151-, the H147A-, and the H. influenzae peptide-encoding viruses exhibited CD4 + Th1 responses to both endogenous viral peptides and to the virus-encoded PLP139–151 peptide by 14 days post-infection (Figure 3A and 3B). Both virus and PLP139–151-specific responses persisted until at least 60–90 days post-infection. These results indicate that the PLP139–151 and mimic peptides can be processed from the 30mer insertion within the virus leader polyprotein and that the early onset demyelinating disease temporally correlates with the induction of the autoreactive response to the native PLP139–151 peptide. Interestingly, PLP178– 191-specific T cell responses are observed at 60–90 days post-infection in mice which developed earlyonset disease following infection with either PLP139TMEV, H147A-TMEV or the H. influenzae-TMEV mimic indicating that sustained tissue damage initiated by virus-induced molecular mimicry can subsequently lead to epitope spreading to additional PLP epitopes following the release of endogenous epitopes during chronic myelin destruction.

Implications of the TMEV-PLP Molecular Mimicry Model to Virus-Induced Autoimmunity These studies clearly demonstrate that peripheral or intracerebral infection with TMEV expressing 30mers encompassing the encephalitogenic self myelin PLP139–151 epitope or appropriate mimics (H147A PLP139–151 and H. influenzae—compatible at the primary TCR and MHC contact residues) can induce an early-onset immune-mediated demyelinating disease in mice due to the rapid activation of PLP139– 151-specific CD4 + Th1 cells. In contrast, a potential mimic virus expressing a non-conservative amino acid substitution at the primary TCR contact site (W144A PLP139–151) did not induce the early demyelinating

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disease and did not stimulate a cross-reactive CD4 + T cell response to PLP139–151. This in conjunction with the tolerance data provides direct evidence that PLP139–151-specific mimicry is responsible for the initiation of this early-onset virus-induced demyelinating disease. These findings are consistent with earlier studies demonstrating that immunization of SJL mice with minimal PLP139–151 APLs in complete Freund’s adjuvant (CFA) carrying H→L or H→A substitutions at position 147 induce T cell responses cross-reactive wit the native PLP139–151 peptide and are thus encephalitogenic [36, 37], whereas immunization with the W144A in CFA was immunogenic in that it primed a specific CD4 + T cell response, but did not lead to clinical EAE as the T cells failed to cross-react with the native PLP139–151 epitope [34]. It thus appears that to be an effective mimic epitope that close homology must be maintained at the primary TCR contract residue. It is also significant that TMEV expressing encephalitogenic PLP mimic epitopes could induce early-onset disease in the absence of accessory stimuli associated with complete Freund’s adjuvant indicating that TMEV can activate sufficient innate immune signals to lead to the activation of the autoreactive T cell population. In support of this, our recent evidence indicates that TMEV infection of quiescent microglia (macrophages and microglia are the primary targets of TMEV infection and persistence [38, 39]) results in upregulation of cytokines involved in the innate immune response (type I interferons, TNF-, IL-6, IL-12, and IL-18) and upregulation of MHC class II and co-stimulatory molecules (B7-1, B7-2, CD40 and ICAM-1) which enabled the microglia to efficiently process and present both virus and myelin antigens to inflammatory CD4 + Th1 cells (manuscript submitted). The extremely efficient ‘adjuvanticity’ of TMEV is also illustrated by the fact that early onset disease was induced by i.c. infection of SJL mice with TMEV encoding the H. influenzae mimic peptide (Table 1). In contrast, it has been reported [36] and we have confirmed that multiple immunizations of SJL mice with this minimal peptide in CFA failed to induce clinical EAE. As shown in Figure 4, immunization of SJL with the minimal H. influenzae peptide in CFA results in minimal differentiation of IFN--producing Th1 cells cross-reactive with the native PLP139–151 epitope and therefore does not lead to clinical disease. In contrast, infection of SJL mice with TMEV encoding the H. influenzae mimic peptide leads to the induction of high IFN--producing Th1 cells cross-reactive with PLP139–151 which can initiate clinical disease. The type of virus and the primary host cell type infected by a virus encoding a potential self mimic epitope appear to be important variables in determining whether an autoimmune response/disease is actually induced. It has been reported that infection of SJL mice with a vaccinia virus construct expressing the entire coding region of PLP failed to induce overt CNS disease, but rendered mice more susceptible to later induction of PLP139–151-induced EAE [40]. Further, infection of mice with a vaccinia construct encoding an encephalitogenic MBP peptide actually

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Figure 4. Proposed mechanism for expansion of the cross-reactive T cell repertoire following infection with TMEV encoding the Haemophilus influenzae-derived molecular mimic peptide of the immunodominant encephalitogenic PLP139–151 epitope. See text for details.

protected mice from later attempts to induce EAE via MBP peptide/CFA immunization [41]. Vaccinia infects many cell types including B7 costimulatory moleculedeficient B cells which may result in inefficient or incomplete priming of autoantigen-specific CD4 + T cells or, in some cases, actually induce unresponsiveness in autoreactive T cells. Thus, it appears that depending on the nature of the environment in which a mimic epitope is presented to a potentially autoreactive T cell, a full spectrum of possible receptormediated outcomes is possible ranging from full T cell activation to T cell antagonism.

Summary Studies in our laboratory employing infection of SJL mice with wild-type TMEV and TMEV encoding molecular mimics of the encephalitogenic PLP139–151 peptide clearly demonstrate that both epitope spreading and molecular mimicry are viable mechanisms to explain the induction of autoreactive T cells following virus infection. These studies also indicate that these mechanisms are not mutually exclusive as induction of early onset disease with a PLP139–151 mimic expressing strain of TMEV can lead to epitope spreading to the non-crossreactive PLP178–191 epitope later in disease (Table 1). This unique model system should allow the identification of disease-associated mimic epitopes capable of being processed from their native sequences, and their potential involvement in the

pathogenesis of MS and other autoimmune diseases by employing the appropriate HLA class II transgenic mice [42].

Acknowledgements This work was supported in part by USPHS NIH Grants NS23349 and NS40460 and National Multiple Sclerosis Society Grant 3166-A-4.

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