The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of ...virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC50 value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.
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•Characterization of potent human monoclonal SARS-CoV-2-neutralizing antibodies•Some SARS-CoV-2 antibodies reacted with mammalian self-antigens in different organs•Crystal structures of two antibodies in complex with SARS-CoV-2 RBD at 2.55/2.70 Å•Post-exposure antibody treatment protected from lung damage in infected hamsters
Kreye et al. report isolation and characterization of monoclonal antibodies from COVID-19 patients, some of which were found to display autoreactivity with mammalian self-antigens in different organs. Crystal structures of two antibodies in complex with the SARS-CoV-2 spike RBD reveal antibody engagement with the ACE2 binding site from different approach angles. One antibody was evaluated further for in vivo efficacy and found to be both protective and efficacious post-challenge in a hamster infection model.
Objective
Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely ...understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross‐reactivity.
Methods
IgG‐secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full‐length mAb (n = 93) and also as germline‐reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated.
Results
Four unrelated anti–citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell–derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay.
Conclusion
These findings suggest that the high level of cross‐reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.
Objective
Anti–citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease‐relevant autoantigens that are targeted by ACPAs remains a ...matter of debate. This study utilized patient‐derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA.
Methods
Twelve human monoclonal ACPAs (positive by the second‐generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head‐to‐head comparison of their reactivities was performed. For profiling, we used a complementary DNA–based protein array (Engine GmbH) and 3 peptide‐screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone‐derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products).
Results
The fine‐specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone‐distinct consensus binding motifs (e.g., Cit–Gly, Gly–Cit, or Arg–Cit–Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA‐binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides.
Conclusion
ACPAs and anti–modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti–carbamylated and anti–citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.
Abstract
Background
Rheumatoid arthritis (RA) is classified as seropositive or seronegative, depending on the presence/absence of rheumatoid factor (RF), primarily IgM RF, and/or anti-citrullinated ...protein antibodies (ACPA), commonly detected using anti-cyclic citrullinated peptide (CCP) assays. Known risk factors associate with the more severe seropositive form of RA; less is known about seronegative RA. Here, we examine risk factors and clinical phenotypes in relation to presence of autoantibodies in the RA subset that is traditionally defined as seronegative.
Methods
Anti-CCP2 IgG, 19 ACPA fine-specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies, were analysed in 2755 RA patients and 370 controls. Antibody prevalence, levels, and co-occurrence were examined, and associations with risk factors and disease activity during 5 years were investigated for different antibody-defined RA subsets.
Results
Autoantibodies were detected in a substantial proportion of the traditionally defined seronegative RA subset, with ACPA fine-specificities found in 30%, IgA/IgG RF in 9.4%, and anti-CarP antibodies in 16%, with a 9.6% co-occurrence of at least two types of RA-associated autoantibodies. HLA-DRB1 shared epitope (SE) associated with the presence of ACPA in anti-CCP2-negative RA; in anti-CCP2-positive RA, the SE association was defined by six ACPA fine-specificities with high co-occurrence. Smoking associated with RF, but not with ACPA, in anti-CCP2-negative RA. Presence of ACPA and RF, but not anti-CarP antibodies, in conventionally defined “seronegative” RA, associated with worse clinical outcome.
Conclusions
“Seronegative” RA is not truly a seronegative disease subset. Additional screening for ACPA fine-specificities and IgA/IgG RF defines a group of patients that resembles seropositive patients with respect to risk factors and clinical picture and may contribute to earlier diagnosis for a subset of anti-CCP2−/IgM RF− patients with a high need for active treatment.
Objective
Anti–citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B ...cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen–specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity.
Methods
Blood samples were obtained from patients in a University of Minnesota cohort with ACPA‐positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α‐enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer–bound B cells were subjected to flow cytometric cell sorting and single‐cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality.
Results
Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V‐region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline‐dependent cross‐reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen–specific mAb with cross‐reactive binding profiles also promoted arthritis in mice.
Conclusion
Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline‐multispecific B cell clades with pathogenic potential.
Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, ...yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.
Abstract
Background
There is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the ...hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.
Methods
Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry.
Results
HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CN
DL
-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CN
DL
-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CN
DL
-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.
Conclusion
CN
DL
-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter ...quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J ...rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the "Golden Agers".
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