Serum IgG Profiling of Toddlers Reveals a Subgroup with Elevated Seropositive Antibodies to Viruses Correlating with Increased Vaccine and Autoantigen Responses.

PURPOSE: The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. METHODS: An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. RESULTS: IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma. CONCLUSION: Serum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease.

Soluble antigen arrays improve the efficacy and safety of peptide-based tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but the limited in vivo stability and uptake of peptides impedes clinical implementation. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Both SAgA variants significantly delayed anaphylaxis compared to their respective free peptides. The anaphylaxis, which occurred in NOD mice but not in C57BL/6 mice, was dose-dependent but did not correlate with the production of IgG1 or IgE against the peptides. We provide evidence that SAgAs significantly improve the efficacy and safety of peptide-based immunotherapy.

Complement and myasthenia gravis.

Myasthenia gravis is a neuromuscular disease associated with antibodies against components of the neuromuscular junction, most often against the acetylcholine receptor (AChR). Although several mechanisms have been postulated to explain how these autoantibodies can lead to the pathology of the disease, convincing evidence suggests that destruction of the receptor-bearing postsynaptic membrane by complement membrane attack complex is of central importance. In this review, evidence for the importance of complement, and possible relationships between autoantigen, autoantibodies, complement activation, and the destruction of the membrane are discussed. More recent insights from the results of the complement-inhibiting therapeutic antibody eculizumab are also described, and the mechanisms connecting antibody binding to complement activation are considered from a structural viewpoint.

Association of serum anti-centromere protein F antibodies with clinical response to infliximab in patients with rheumatoid arthritis: A prospective study.

BACKGROUND: One-third of rheumatoid arthritis (RA) patients demonstrate no clinical improvement after receiving tumor necrosis factor inhibitors (TNFi). The presence of serum autoantibodies is a hallmark in RA and may provide information on future response to treatment. The aim of this prospective study was to search for novel serum autoantibodies useful to predict clinical response to TNFi. METHODS: The autoantibody repertoire was profiled on RA patients treated with TNFi as a first line of biologic therapy (N = 185), who were recruited in three independent cohorts. The presence and levels of autoantibodies in serum at baseline were analysed in association with the clinical response after 24 weeks follow-up. A multiplex bead array built using antigens selected from an initial untargeted screening was employed to identify the autoantibodies on a discovery cohort (N = 50) and to verify and validate the results on verification (N = 61) and validation (N = 74) cohorts. Non-parametric tests, meta-analysis and Receiver Operating Curves (ROC) were performed in order to assess the clinical relevance of the observed findings. RESULTS: Novel autoantibodies were associated with the clinical response to TNFi, showing different reactivity profiles among the different TNFi. The baseline levels of IgG antibodies against Centromere protein F (CENPF), a protein related to cell proliferation, were significantly (p<0.05) increased in responders (N = 111) to infliximab (IFX) compared to non-responders (N = 44). The addition of anti-CENPF antibodies to demographic and clinical variables (age, sex, DAS28-ESR) resulted in the best model to discriminate responders, showing an area under the curve (AUC) of 0.756 (95% CI [0.639-0.874], p = 0.001). A further meta-analysis demonstrated the significant association of anti-CENPF levels with the patient's subsequent response to IFX, showing a standardized mean difference (SMD) of -0.65 (95% CI [-1.02;-0. 27], p = 0.018). CONCLUSIONS: Our study reveals for the first time the potential of circulating anti-CENPF antibodies to predict the clinical response to IFX before starting the treatment. This finding could be potentially useful to guide therapeutic decisions and may lead to further studies focusing on the role of CENPF on RA pathology.

Array-Based Profiling of Proteins and Autoantibody Repertoires in CSF.

Protein profiling enabled through affinity proteomics represents a powerful strategy for analysis of complex samples such as human body fluids. Cerebrospinal fluid (CSF) is the proximal fluid of the central nervous system and is commonly analyzed in the context of neurological diseases. Through the presence of brain-derived proteins, this fluid can offer insight into the physiological state of the brain. Here, we describe multiplex and flexible protein and autoantibody profiling approaches using suspension bead arrays. Through minimal sample processing, these methods enable high-throughput analysis of hundreds of samples and proteins in one single assay and thereby provide powerful approaches for discovery of disease-associated proteins and autoantigens.

Multiplexed Antigen Bead Arrays for the Assessment of Antibody Selectivity and Epitope Mapping.

With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.

Protein Arrays II: Antigen Arrays.

Antigen arrays are fabricated using various antigens such as DNA, histones, synthetic peptides, recombinant proteins, or cell extracts to detect autoantibodies in autoimmune diseases, alloantibodies in transplantation, drug-induced antibodies or cancer-induced antibodies in blood or cell culture supernatant. In this protocol, we will provide a step-by-step executable procedure to perform antigen arrays, including antigen preparation and printing, blocking, sample loading, array detection, imaging, and data analysis.