Epstein Barr virus nuclear antigen 1 (EBNA-1) peptides recognized by adult multiple sclerosis patient sera induce neurologic symptoms in a murine model.

Multiple sclerosis (MS) is an autoimmune demyelinating disease with progressive neurodegeneration and complex etiology likely involving genetic and environmental factors. MS has been associated with Epstein Barr virus (EBV) infection, with patients often showing enhanced responses to EBV antigens. To determine whether abnormal EBV nuclear antigen-1 (EBNA-1) humoral immunity can serve as an initiator of autoimmune responses in MS, we investigated the fine specificities of the humoral immune response against EBNA-1 in MS patients using solid phase epitope mapping. Antibodies from MS patients recognized an EBNA-1 epitope spanning amino acids 411-426, previously unknown to be recognized specifically by untreated MS patients. Antibodies against this epitope cross-reacted to myelin basic protein (MBP). Furthermore, animals immunized with this EBNA-1 polypeptide mounted a response against MBP and developed signs of experimental autoimmune encephalitis (EAE). These data support a link between MS and EBV through antibodies that cross-react between EBV proteins and the MBP autoantigen.

Clostridium difficile 027/BI/NAP1 encodes a hypertoxic and antigenically variable form of TcdB.

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.

Ribosomal P autoantibodies are present before SLE onset and are directed against non-C-terminal peptides.

Autoantibodies to ribosomal P (ribo P) are found in 15-30% of systemic lupus erythematosus (SLE) patients and are highly specific for SLE. The goal of this study is to assess the temporal association of anti-ribosomal P (anti-P) responses with SLE disease onset, as well as to characterize select humoral ribo P epitopes targeted in early, pre-diagnostic SLE samples. Patients with stored serial serum samples available prior to SLE diagnosis were identified from a military cohort. Each sample was tested for antibodies against ribo P utilizing standard C terminus ribo P enzyme-linked immunosorbent assays (ELISA) and a solid phase, bead-based assay with affinity-purified ribo P proteins. In this study, antibodies to ribo P were more common in African American SLE patients (p = 0.026), and anti-P-positive patients comprised a group with more measured autoantibody specificities than did other SLE patients (3.5 vs 2.2, p < 0.05). Antibodies against ribo P were present on average 1.7 years before SLE diagnosis and were detected an average of 1.08 years earlier in pre-diagnostic SLE samples using affinity-purified whole protein rather than C-terminal peptide alone (p = 0.0019). Furthermore, 61% of anti-P-positive patients initially had antibodies to aa 99-113, a known ribosomal P0 antigenic target, at a time point when no antibodies to the clinically used C terminus were detected. Our findings provide evidence that antibodies against ribosomal P frequently develop before clinical SLE diagnosis and are more broadly reactive than previously thought by targeting regions outside of the C terminus.

60 kD Ro and nRNP A frequently initiate human lupus autoimmunity.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns.

Clinical criteria for systemic lupus erythematosus precede diagnosis, and associated autoantibodies are present before clinical symptoms.

OBJECTIVE: Specific events that occur during the development of systemic lupus erythematosus (SLE) can be quite variable among individual patients. The aim of this study was to identify patterns that distinguish early clinical events in SLE and to assess whether the presence of associated autoantibodies precedes the fulfillment of clinical criteria. METHODS: Through a retrospective chart review of military medical records, 130 patients who met the American College of Rheumatology (ACR) criteria for the classification of SLE were identified. The initial time at which each criterion was fulfilled was recorded. Autoantibody analysis was performed on serum samples, using enzyme-linked immunosorbent assays or immunofluorescence. RESULTS: The clinical features that were observed earliest were discoid rash and seizures, which developed a mean 1.74 and 1.70 years, respectively, before the diagnosis of SLE; however, arthritis was the criterion that was most commonly observed before diagnosis. The presence of IgG rheumatoid factor (IgG-RF) preceded the development of arthritis in 15 (94%) of the 16 patients who were positive for IgG-RF and in whom arthritis developed (Z = 10.2, P < 0.0001). Analogously, IgM-RF appeared before the development of arthritis in 13 (76%) of 17 patients. Anti-double-stranded DNA antibodies were associated with renal disease and appeared before evidence of nephritis in most patients (92%) (Z = 13.3, P < 0.0001). An analysis of the appearance of autoantibodies compared with the appearance of clinical criteria not associated with them revealed no significant temporal relationship. CONCLUSION: Symptoms associated with the ACR criteria for classification of SLE are commonly present before the diagnosis of SLE, and development of organ-associated autoantibodies generally precedes the appearance of their associated clinical features.

Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry.

The origins of autoimmunity in systemic lupus erythematosus (SLE) are thought to involve both genetic and environmental factors. To identify environmental agents that could potentially incite autoimmunity, we have traced the autoantibody response in human SLE back in time, prior to clinical disease onset, and identified the initial autoantigenic epitope for some lupus patients positive for antibodies to 60 kDa Ro. This initial epitope directly cross-reacts with a peptide from the latent viral protein Epstein-Barr virus nuclear antigen-1 (EBNA-1). Animals immunized with either the first epitope of 60 kDa Ro or the cross-reactive EBNA-1 epitope progressively develop autoantibodies binding multiple epitopes of Ro and spliceosomal autoantigens. They eventually acquire clinical symptoms of lupus such as leukopenia, thrombocytopenia and renal dysfunction. These data support the hypothesis that some humoral autoimmunity in human lupus arises through molecular mimicry between EBNA-1 and lupus autoantigens and provide further evidence to suspect an etiologic role for Epstein-Barr virus in SLE.

The prevalence, onset, and clinical significance of antiphospholipid antibodies prior to diagnosis of systemic lupus erythematosus.

OBJECTIVE: To determine whether antiphospholipid antibodies (aPL) occur before the diagnosis of systemic lupus erythematosus (SLE) and before initial clotting events, and whether their presence early in the disease course influences clinical outcome. METHODS: Serum samples obtained from 130 lupus patients before and after SLE diagnosis were screened for IgG and IgM aPL using an anticardiolipin (aCL) enzyme-linked immunosorbent assay. Medical records of all patients were carefully reviewed for data on the time of onset of SLE features meeting clinical criteria and on disease manifestations. RESULTS: Twenty-four patients (18.5%) were positive for IgG and/or IgM aCL prior to SLE diagnosis. Anticardiolipin antibodies appeared from 7.6 years prior to SLE diagnosis to within the same month as SLE diagnosis, with a mean onset occurring 3.0 years before SLE diagnosis. Additionally, aCL presence early in the disease process seemed to predict a more severe clinical outcome; these patients eventually met an average of 6.1 of the 11 classification criteria for SLE, compared with 4.9 criteria for other patients (P < 0.001). The early aCL-positive population also had more frequent renal disease, central nervous system disease, thrombocytopenia, and clotting events. In this population, aCL preceded initial thrombotic events by a mean of 3.1 years. CONCLUSION: Anticardiolipin antibodies in SLE patients tend to precede initial clotting events by several years. Furthermore, the presence of early, prediagnosis aPL seems to herald a more varied, severe clinical course with earlier onset in patients with SLE.