Sterile protection against relapsing malaria with a single-shot vaccine.

Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria. Such an approach has not been fully evaluated for relapsing malaria. Here, in the P. cynomolgi-rhesus macaque model for relapsing malaria, we employ the parasites' natural relapsing phenotype to self-boost the immune response against liver-stage parasites, following a single-shot high-dose live sporozoite vaccination. This approach resulted in sterile protection against homologous sporozoite challenge in three out of four animals in the group that was also exposed for several days to blood stages during primary infection and relapses. One out of four animals in the group that received continuous chemoprophylaxis to abort blood-stage exposure was also protected from sporozoite challenge. Although obtained in a small number of animals as part of a Proof-of-Concept study, these results suggest that limited blood-stage parasite exposure may augment protection in this model. We anticipate our data are a starting point for further research into correlates of protection and extrapolation of the single-shot approach to develop efficacious malaria vaccines against relapsing human malaria.

A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits.

BACKGROUND: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. METHOD: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. RESULTS: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. DISCUSSION: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

Amyloid-like Behavior of Site-Specifically Citrullinated Myelin Oligodendrocyte Protein (MOG) Peptide Fragments inside EBV-Infected B-Cells Influences Their Cytotoxicity and Autoimmunogenicity.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34-56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35-55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein.

EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E-restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20(+) spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40-48 core epitope. Finally, LCV infection also induced expression of LC3-II(+) cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40-48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40-48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs.

Immune profile of an atypical EAE model in marmoset monkeys immunized with recombinant human myelin oligodendrocyte glycoprotein in incomplete Freund's adjuvant.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) in the common marmoset monkey (Callithrix jacchus) is a relevant preclinical model for translational research into immunopathogenic mechanisms operating in multiple sclerosis (MS). Prior studies showed a core pathogenic role of T and B cells specific for myelin oligodendrocyte glycoprotein (MOG). However, in those studies, the quality of the response against MOG epitopes was strongly biased by bacterial antigens in the complete Freund's adjuvant (CFA), in which the immunizing recombinant human (rh) MOG protein had been formulated. In response to the need of a more refined EAE model, we have tested whether disease could also be induced with rhMOG in incomplete Freund's adjuvant (IFA). METHOD: Marmosets were immunized with rhMOG emulsified in IFA in the dorsal skin. Monkeys that did not develop neurological deficit were given booster immunizations at 28-day interval with the same antigen preparation. In a second experiment, three marmoset twin pairs were sensitized against MOG peptides in IFA to study a possibility for suppressive activity towards pathogenic T cells directed against the encephalitogenic epitope MOG40-48. RESULTS: Despite the absence of strong danger signals in the rhMOG/IFA inoculum, all monkeys developed clinically evident EAE symptoms. Moreover, in all monkeys, demyelinated lesions were present in the white matter and in two cases also in the cortical grey matter. Immune profiling at height of the disease showed a dominant T cell response against the overlapping peptides 14-36 and 24-46, but reactivity against the pathogenically most relevant peptide 34-56 was conspicuously absent. In the second experiment, there was an indication for a possible suppressive mechanism. CONCLUSIONS: Immunization of marmoset monkeys with rhMOG in IFA elicits clinical EAE in all animals. Moreover, rhMOG contains pathogenic and regulatory epitopes, but the pathogenic hierarchy of rhMOG epitopes is strongly influenced by the adjuvant in which the protein is formulated.

Induction of experimental autoimmune encephalomyelitis with recombinant human myelin oligodendrocyte glycoprotein in incomplete Freund's adjuvant in three non-human primate species.

The experimental autoimmune encephalitis (EAE) model is used for preclinical research into the pathogenesis of multiple sclerosis (MS), mostly in inbred, specific pathogen free (SPF)-raised laboratory mice. However, the naive state of the laboratory mouse immune system is considered a major hurdle in the translation of principles from the EAE model to the MS patient. Non-human primates (NHP) have an immune system harboring T- and B-cell memory against environmental antigens, similar as in humans. We sought to further refine existing NHP EAE models, which may help to bridge the gab between mouse EAE models and MS. We report here on new EAE models in three NHP species: rhesus monkeys (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis) and common marmosets (Callithrix jacchus). EAE was induced with recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG) formulated in incomplete Freund's adjuvant (IFA). IFA lacks the bacterial antigens that are present in complete Freund's adjuvant (CFA), which are notorious for the induction of discomforting side effects. Clinically evident EAE could be induced in two out of five rhesus monkeys, six out of six cynomolgus monkeys and six out of six common marmosets. In each of these species, the presence of an early, high anti-rhMOG IgM response is correlated with EAE with an earlier onset and more severe disease course. Animals without an early high IgM response either did not develop disease (rhesus monkeys) or developed only mild signs of neurological deficit (marmoset and cynomolgus monkeys).

The different clinical effects of anti-BLyS, anti-APRIL and anti-CD20 antibodies point at a critical pathogenic role of γ-herpesvirus infected B cells in the marmoset EAE model.

The robust and rapid clinical effect of depleting anti-CD20 monoclonal antibodies (mAb) in multiple sclerosis (MS) demonstrates a critical pathogenic contribution of B cells. The clinical effect of anti-CD20 mAb has been replicated in a relevant preclinical MS model, experimental autoimmune encephalomyelitis (EAE) in marmoset monkeys (Callithrix jacchus). By contrast, treatment with mAbs against two essential cytokines in B cell activation growth and survival, i.e. BlyS/BAFF and APRIL, was only partially effective. All three mAbs induced depletion of CD20+ B cells from the circulation, albeit with different kinetics and based on distinct mechanisms of action. In the current study we analyzed whether the different clinical effect of anti-CD20 mAb or the anti-BLyS and anti-APRIL mAbs is due to different depletion of B cells infected with the EBV of marmosets, CalHV3. Employing a novel PCR-based assay, half of the colony of group-housed marmosets was tested positive for CalHV3 DNA in secondary lymphoid organs. The same prevalence was observed in placebo-treated monkeys. In marmosets treated with anti-CD20 mAb the load of CalHV3 DNA in lymphoid organs was substantially reduced, while this was not observed in the monkeys treated with anti-BLyS or anti-APRIL mAbs. To examine the pathogenic role of virus-transformed B cells, we infused EBV-transformed B lymphoblastic cell (BLC) lines presenting the immunodominant MOG34-56 peptide. We observed in the recipients of MOG34-56 pulsed BLC, but not in their fraternal siblings infused with non-pulsed BLC, activation of anti-MOG34-56 T cells and meningeal inflammation. Collectively, the data show that among CD20+ B cells, the herpesvirus-transformed subset has a particularly important pathogenic role in the marmoset EAE model.

Antibodies against human BLyS and APRIL attenuate EAE development in marmoset monkeys.

B lymphocyte stimulator (BLyS, also indicated as BAFF (B-cell activating factor) and CD257), and A Proliferation Inducing Ligand (APRIL, CD256) are two members of the TNF superfamily with a central role in B cell survival. Antibodies against these factors have potential therapeutic relevance in autoimmune inflammatory disorders with a proven pathogenic contribution of B cells, such as multiple sclerosis (MS). In the current study we performed a multi-parameter efficacy comparison of monoclonal antibodies against human anti-BLyS and anti-APRIL in a common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE). A MS-like disease was induced by immunization with recombinant human myelin/oligodendrocyte glycoprotein (rhMOG) in complete Freund's adjuvant. The results show that the anti-BLyS and anti-APRIL antibody cause significant depletion of circulating CD20+ B cells, but a small subset of CD20 + CD40(high) B cells was not depleted. Induction of CD20+ B cell depletion from lymph nodes was only observed in the anti-BLyS treated monkeys. Both antibodies had a significant inhibitory effect on disease development, but all monkeys developed clinically evident EAE. Anti-BLyS treated monkeys were sacrificed with the same clinical signs as saline-treated monkeys, but nevertheless displayed significantly reduced spinal cord demyelination. This effect was not observed in the anti-APRIL treated monkeys. The two antibodies had a different effect on T cell subset activation and the profiles of ex vivo released cytokines. In conclusion, treatment with anti-BLyS and anti-APRIL delays the development of neurological disease in a relevant preclinical model of MS. The two mAbs achieve this effect via different mechanisms.

B-cell depletion abrogates T cell-mediated demyelination in an antibody-nondependent common marmoset experimental autoimmune encephalomyelitis model.

CD20-positive B-cell depletion is a highly promising treatment for multiple sclerosis (MS), but the mechanisms underlying therapeutic effects are poorly understood. B cells are thought to contribute to MS pathogenesis by producing autoantibodies that amplify demyelination via opsonization of myelin. To analyze autoantibody-nondependent functions of B cells in an animal model of MS, we used a novel T cell-driven experimental autoimmune encephalomyelitis (EAE) model in marmoset monkeys (Callithrix jacchus). In this model, demyelination of brain and spinal cord white and gray matter and the ensuing neurological deficits are induced by immunization with peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) in incomplete Freund's adjuvant. Although autoantibodies do not have a detectable pathogeniccontribution in the model, depletion of B cells with monoclonal antibody 7D8, a human IgG1κ monoclonal antibody against human CD20, suppressed clinical and pathological EAE. In B cell-depleted monkeys, the activation of peptide-specific Th17-producing and cytotoxic T cells, which in previous studies were found to play an essential role in disease induction, was impaired. Thus, we demonstrate a critical antibody-nondependent role for B cells in EAE, that is, the activation of pathogenic T cells.

Discrepant effects of human interferon-gamma on clinical and immunological disease parameters in a novel marmoset model for multiple sclerosis.

The core pathogenic process in the common marmoset model of multiple sclerosis (MS) is the activation of memory-like T cells specific for peptide 34 to 56 derived from the extracellular domain of myelin/oligodendrocyte glycoprotein (MOG(34-56)). Immunization with MOG(34-56) in incomplete Freund's adjuvant is a sufficient stimulus for in vivo activation of these T cells, together with the induction of MS-like disease and CNS pathology. Ex vivo functional characteristics of MOG(34-56) specific T cells are specific cytolysis of peptide pulsed target cells and high IL-17A production. To indentify possible functions in this new model of T helper 1 cells, which play a central pathogenic role in MS models induced with complete Freund's adjuvant, we tested the effect of human interferon-γ (IFNγ) administration during disease initiation of the disease (day 0-25) and around the time of disease expression (psd 56-81). The results show a clear modulatory effect of early IFNγ treatment on humoral and cellular autoimmune parameters, but no generalized mitigating effect on the disease course. These results argue against a prominent pathogenic role of T helper 1 cells in this new marmoset EAE model.

Unravelling the T-cell-mediated autoimmune attack on CNS myelin in a new primate EAE model induced with MOG34-56 peptide in incomplete adjuvant.

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein (MOG(34-56) ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG(34-56) displayed high production of IL-17A and specific lysis of MOG(34-56) -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG(40-48) presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG(34-56,) comprised CD4(+) CD56(+) and CD4(+) CD8(+) CD56(+) T cells. Furthermore, phenotypical analysis showed that CD4(+) CD8(+) CD56(+) T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.

Induction of progressive demyelinating autoimmune encephalomyelitis in common marmoset monkeys using MOG34-56 peptide in incomplete freund adjuvant.

Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Surgical excision of CNS-draining lymph nodes reduces relapse severity in chronic-relapsing experimental autoimmune encephalomyelitis.

Despite lack of classical lymphatic vessels in the central nervous system (CNS), cells and antigens do reach the CNS-draining lymph nodes. These lymph nodes are specialized to mediate mucosal immune tolerance, but can also generate T- and B-cell immunity. Their role in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) therefore remains elusive. We hypothesized that drainage of CNS antigens to the CNS-draining lymph nodes is vital for the recurrent episodes of CNS inflammation. To test this, we surgically removed the superficial cervical lymph nodes, deep cervical lymph nodes, and the lumbar lymph nodes prior to disease induction in three mouse EAE models, representing acute, chronic, and chronic-relapsing EAE. Excision of the CNS-draining lymph nodes in chronic-relapsing EAE reduced and delayed the relapse burden and EAE pathology within the spinal cord, which suggests initiation of CNS antigen-specific immune responses within the CNS-draining lymph nodes. Indeed, superficial cervical lymph nodes from EAE-affected mice demonstrated proliferation against the immunizing peptide, and the deep cervical lymph nodes, lumbar lymph nodes, and spleen demonstrated additional proliferation against other myelin antigen epitopes. This indicates that intermolecular epitope spreading occurs and that CNS antigen-specific immune responses are differentially generated within the different CNS-draining lymphoid organs. Proliferation of splenocytes from lymphadenectomized and sham-operated mice against the immunizing peptide was similar. These data suggest a role for CNS-draining lymph nodes in the induction of detrimental immune responses in EAE relapses, and conclusively demonstrate that the tolerance-inducing capability of cervical lymph nodes is not involved in EAE.

Axonal loss and gray matter pathology as a direct result of autoimmunity to neurofilaments.

Axonal damage is considered the major cause of irreversible disability in multiple sclerosis (MS). Which mechanisms underlie the damage and whether this is secondary to myelin damage remains to be clarified. Recently, we have demonstrated that autoimmunity to the axonal/neuronal cytoskeletal protein neurofilament light (NF-L) induces axonal damage and neurological disease including spasticity - a common feature of MS. To examine the relationship between axonal damage and demyelination we have characterized the detailed neuropathology of NF-L-induced disease in Biozzi mice compared to classical experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein (MOG). In NF-L-induced neurological disease the lesions were predominantly located in the dorsal column displaying extensive axonal degeneration, but were also abundant in the gray matter. In contrast, lesions in MOG-EAE were restricted to the lateral and ventral columns and displayed less axonal damage and little gray matter involvement. The differential lesion location was confirmed by quantitation of leukocyte subsets. In both diseases myelin damage was a common feature although the numerous empty myelin sheaths in NF-L-disease indicative of axonal damage suggest that myelin damage was a secondary event. In summary, autoimmunity to NF-L induces a distinct lesion topology, axonal damage and gray matter lesions supporting the notion that axonal loss and gray matter pathology can be the direct consequence of a primary autoimmune attack against axonal antigens such as NF-L rather than merely a secondary event to myelin damage.

Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice.

Axonal damage is the major cause of irreversible neurologic disability in patients with multiple sclerosis. Although axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we show that Biozzi ABH mice immunized with NF-L protein develop neurologic disease characterized by spastic paresis and paralysis concomitant with axonal degeneration and inflammation primarily in the dorsal column of the spinal cord. The inflammatory central nervous system lesions were dominated by F4/80+ macrophages/microglia and relatively low numbers of CD4+ and CD8+ T-cells. In splenocyte cultures, proliferation to NF-L was observed in CD4+ T-cells accompanied by the production of the proinflammatory cytokine interferon-gamma. Elevated levels of circulating antibodies recognizing recombinant mouse NF-L were present in the serum, and immunoglobulin deposits were observed within axons in spinal cord lesions of mice exhibiting clinical disease. These data provide evidence that autoimmunity to NF-L protein induces axonal degeneration and clinical neurologic disease in mice, indicating that autoimmunity to axonal antigens, as described in multiple sclerosis, may be pathogenic rather than acting merely as a surrogate marker for axonal degeneration.

Encephalitogenic and tolerogenic potential of altered peptide ligands of MOG and PLP in Biozzi ABH mice.

Altered peptide ligands (APL) are highly effective in inhibiting experimental autoimmune encephalomyelitis (EAE) in rodents although clinical trials in multiple sclerosis reveal severe limitations probably due to the diverse and differential effects of APL in vivo compared to in vitro. Myelin oligodendrocyte glycoprotein (MOG 8-21) induces relapsing EAE in ABH (A(g7)) mice associated with broadening of the autoimmune repertoire thus providing a dynamic system to examine the efficacy of peptide analogues. Subtle changes in MOG 8-21 dramatically influenced disease susceptibility and T cell responses in vitro. Non-encephalitogenic APL that induce production of the 'regulatory' cytokines IL-10 and/or TGFbeta and concomitant low levels of the 'proinflammatory' cytokines IFNgamma and TNFalpha modulated relapsing EAE but were far less effective than the 'proinflammatory' wild-type MOG 8-21 peptide. These data reveal that APL differ greatly in their ability to activate encephalitogenic T cells. The extensive heterogeneity of responses of APL in vitro suggests that selection of APL on this criteria is highly unpredictable and probably less effective for therapy than selecting the dominant wild-type epitope and delivering it using a tolerogenic route.

Epitope spread is not critical for the relapse and progression of MOG 8-21 induced EAE in Biozzi ABH mice.

Emerging autoimmunity (epitope-spreading) generated as a consequence of myelin damage is suggested to underlie the relapses in multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG 8-21) induces relapsing EAE in ABH mice characterized by broadening of the autoimmune reportoire. Despite epitope spreading tolerance to the priming antigen, but not emerging epitope reactivities, resulted in long-term inhibition of clinical relapse. In contrast, spinal cord homogenate induced EAE was dominated by a proteolipid protein (PLP 56-70) autoreactivity despite the plethora of CNS antigens in the immunogen. This data suggests that during relapsing-remitting demyelinating disease the pathogenic process is dominated by the initiating antigen, with only a minor role played by emerging T-cell populations. These findings may have important implications for the efficacy of antigen-based immune therapies in autoimmune disorders.

Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice.

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein fails to fully address the possible pathogenic role of the full-length myelin-derived protein expressing post-translational modifications. Immunization of mice with central nervous system tissues from wild-type (WT) and MOG-deficient (MOG(-/-)) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice. While immunization with WT spinal cord homogenate (SCH) resulted in a progressive EAE phenotype, MOG(-/-) SCH induced a mild self-limiting acute disease. Following acute EAE with MOG(-/-) SCH, mice developed T cell responses to recombinant mouse MOG (rmMOG), indicating that MOG released from myelin is antigenic; however, the lack of chronic disease indicates that such responses were not pathogenic. Chronic demyelinating EAE was observed when MOG(-/-) SCH was reconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total white matter-derived protein). These data reveal that while immunization with the full-length post-translational modified form of MOG in myelin promotes the development of a more chronic autoimmune demyelinating neurological disease, MOG (and/or other myelin proteins) released from myelin during ongoing disease do not induce destructive autoimmunity.

Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.

Neurological deficit in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) is probably a consequence of synergy between T and B cell responses to CNS antigens. During the demyelinating phase of chronic relapsing EAE in ABH mice, anti-myelin oligodendrocyte glycoprotein (MOG) responses were increased compared to the inflammatory acute phase, but such levels did not correlate with the severity of clinical disease. The pathogenicity of antibodies (Ab) to MOG, myelin basic protein (MBP), proteolipid protein (PLP) and galactocerebroside (GalC) was investigated in vivo following injection at the onset of EAE. An IgG2a monoclonal Ab (mAb), clone Z12, directed to MOG augmented clinical disease and demyelination in ABH and C57BL/6 mice, but not MOG knock-out mice. No effect was observed with F(ab(2))' fragments of Z12 or with the anti-MOG IgG1 mAbs, clones Y10 or 8-18C5. Cobra venom factor partially reduced the augmenting effect of mAb Z12 suggesting a role for complement. The pathogenic effect of anti-myelin Abs was not restricted to MOG since an anti-GalC mAb exacerbated inflammation in the CNS while an MBP mAb (clone 22) reduced clinical disease. Taken together, these data provide further evidence that myelin-reactive Abs generated during autoimmune neurological disease may play an important role not only in the pathogenesis of disease but also the regulation of myelin-targeted autoimmune disease.