Faculty Bibliography

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.

BACKGROUND:Systemic lupus erythematosus (SLE) patients experience a premature and more severe presentation of coronary artery disease. The underlying mechanisms of accelerated coronary artery disease in SLE patients remain to be elucidated. METHODS:By using atherosclerosis combining a SLE murine model, we proved that the onset of SLE aggravates atherosclerosis. Although the onset of SLE reduced blood lipids slightly, immune deviation contributed to aggravated atherosclerosis in lupus mice. Lupus atheroma were characterized by inflammatory cell infiltration, such as gathered dendritic cells, macrophages, and IgG deposition. RESULTS:Decreased lymphocytes and magnified dendritic cells in the spleen were also observed in lupus mice. Hydroxychloroquine prevented atherosclerosis progression mainly by reversing immune status abnormality caused by SLE. Serum interferon alfa levels were not changed in lupus mice. CONCLUSION/CONCLUSIONS:These findings strongly suggested that anti-inflammatory therapies and hydroxychloroquine provide a new possible strategy for treating SLE patients with atherosclerosis.

Throughout our lives we are immersed in, and colonized by, immense and complex microbial communities. These microbiota serve as activators and early sparring partners for the progressive construction of the layers within our immune defenses and are essential to immune homeostasis. Yet, at times imbalances within the microbiota may contribute to metabolic and immune regulatory abnormalities that underlie the development of inflammatory and autoimmune diseases. Here, we review recent progress in investigations of the microbiome, with emphasis on the gut microbiota associated with systemic autoimmunity. In particular, these studies are beginning to illuminate aspects of the pathogenesis of Systemic Lupus Erythematosus, and may suggest that interconnections with specific disease-associated patterns of dysbiosis within gut communities are bidirectional and mutually reinforcing.

Background/Purpose : Systemic lupus erythematosus (SLE) is the archetypic systemic autoimmune disease, for which there is mounting evidence for roles for intestinal bacteria in the development of the systemic autoimmune responses, inflammation, and tissue injury. Whereas Lupus nephritis (LN) is amongst the most serious and potentially life-threatening complications of SLE, earlier diagnosis would speed clinical decision-making and initiation of definitive therapy. To characterize the interrelationships between Lupus host immunity and a candidate gut microbiome pathobiont, we have recently described an assay for detection of serum IgG responses to the commensal anaerobe, Ruminococcus gnavus ( RG ), for identification of individuals with active LN in three US adult Lupus cohort. In the current studies, we further validated this assay and investigated its potential utility in a Swedish cohort. Methods : Patients were recruited and characterized at the Karolinska Hospital, based on ACR criteria, with 38 LN confirmed by renal biopsy, 38 non-renal SLE patients, with 37 age-gender-ethnicity-matched unaffected population (Healthy) controls, 20 ANCA-associated vasculitis (AAV) and 14 IgA Nephropathy (IgAN) patients, based on renal biopsy and/or standard clinical criteria. At our NYU lab, serologic studies were performed with a previously described custom bead-based serologic assay with nuclease-treated RG2 extract and control analytes. Results : LN patients had significantly higher levels of IgG anti-RG2 than Healthy subjects (p< 0.0001,) other nonrenal Lupus patients (p=0.0002), or patients with IgAN (p< 0.0001) or AAV (p< 0.0001)(Wilcoxon rank-sum). There was a modest difference between non-renal Lupus patients, and Healthy subjects (p=0.0283), but no difference between Healthy with IgAN (p=0.273) or AAV (p=0.433). ROC analysis, when LN were compared to Healthy subjects gave an AUC of 0.863 and a Likelihood ratio (LR) of 5.6 at a cut-offof the mean+1 SD for control subjects (p< 0.0001); the LR was 5.8 for a cut-offof a mean+2SD (p< 0.0062), and LR of 7.8 for a cut-offof a mean+3SD (p=0.0284). For LN compared to the disease controls (AAV and IgAN) there was an AUC of 0.828 with a LR of 2.64 with a cut-offof a mean+1SD for disease controls (p=0.01), and the LR of 5.4 with a disease control mean+2SD (p=0.0071)(Figure 1A&B). Conclusion : These findings further validate that serum IgG anti-RG2 levels provide a robust biomarker for the identification of LN, unaffected by gender and age. Importantly, the assay displayed attractive performance characteristics Area under the curve estimates for comparisons of Lupus nephritis patients to A) age-and gender-matched population (Healthy) controls, or B) Disease controls with AAV or IgA nephropathy that represent other forms of glomerulopathy often complicated by pathologic levels of proteinuria. for discrimination of LN patients from population controls as well as those with pathologic proteinuria due to other forms of immune glomerulopathy (i.e., AAV and IgAN). Our findings also provide circumstantial evidence of a possible role for the R. gnavus pathobiont in patients in Sweden, suggesting this pathobiont may be involved in LN at sites geographically distant from the localities in which this association was first identified. (Figure Presented)

BACKGROUND/PURPOSE/OBJECTIVE:To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity. METHODS:In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts. RESULTS:strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts. CONCLUSION/CONCLUSIONS:These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.