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1.  Clostridium difficile 027/BI/NAP1 Encodes a Hypertoxic and Antigenically Variable Form of TcdB 
PLoS Pathogens  2013;9(8):e1003523.
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.
Author Summary
During the past decade, the C. difficile BI/NAP1/027 strain has emerged and in some settings predominated as the cause of C. difficile infection. Moreover, in some reports C. difficile BI/NAP1/027 has been associated with more severe disease. The reasons for association of this strain with more severe disease and relapse are poorly understood. We compared the toxicity and antigenic profiles of the major C. difficile virulence factor, TcdB, from a previously studied reference strain and a BI/NAP1/027 strain. The results indicate TcdB027, the toxin from the BI/NAP1/027 strain, is more lethal and causes more extensive brain hemorrhaging than TcdB003, the toxin produced by a reference strain of C. difficile. Furthermore, the results show that the antigenic carboxy-terminal domain (CTD) encodes at least 11 epitopes that differ between the two forms of TcdB. In line with this, experiments demonstrate that antiserum against the CTD does not cross-neutralize TcdB003 and TcdB027 toxicity against CHO cells, and TcdB027 appears to be devoid of neutralizing epitopes in this domain. These findings indicate differences in TcdB003 and TcdB027 contribute to increased virulence of C. difficile BI/NAP1/027 and reduce the likelihood of acquired immunity providing cross-protection against infection by these strains.
doi:10.1371/journal.ppat.1003523
PMCID: PMC3731247  PMID: 23935501
2.  Ribosomal P Autoantibodies are Present Before SLE Onset and are Directed Against non-C Terminal Peptides 
Autoantibodies to ribosomal 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 the humoral ribosomal P (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 ELISAs 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.
doi:10.1007/s00109-010-0618-1
PMCID: PMC2877769  PMID: 20396862
lupus; antibodies; autoimmunity; ribosomal P; epitope
3.  60 kD Ro and nRNP A Frequently Initiate Human Lupus Autoimmunity 
PLoS ONE  2010;5(3):e9599.
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.
doi:10.1371/journal.pone.0009599
PMCID: PMC2835743  PMID: 20224770

Results 1-3 (3)