Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation mainly affecting the joints leading to cartilage and bone destruction. The definition of seropositive or seronegative RA is based on the presence or absence of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs). Other autoantibodies have been identified in the last decade such as antibodies directed against carbamylated antigens, peptidyl-arginine deiminase type 4 and v-Raf murine sarcoma viral oncogene homologue B. In order to identify relevant autoantigens, we screened a random peptide library (RPL) with pooled IgGs obtained from 50 patients with seronegative RA. Patients’ sera were then used in an ELISA test to identify the most frequently recognized peptide among those obtained by screening the RPL. Sera from age- and sex-matched healthy subjects were used as controls. We identified a specific peptide (RA-peptide) recognized by RA patients’ sera, but not by healthy subjects or by patients with other immune-mediated diseases. The majority of sera from seronegative and seropositive RA patients (73.8% and 63.6% respectively) contained IgG antibodies directed against the RA-peptide. Interestingly, this peptide shares homology with some self-antigens, such as Protein-tyrosine kinase 2 beta, B cell scaffold protein, Liprin-alfa1 and Cytotoxic T lymphocyte protein 4. Affinity purified anti-RA-peptide antibodies were able to cross react with these autoantigens. In conclusion, we identified a peptide that is recognized by seropositive and, most importantly, by seronegative RA patients’ sera, but not by healthy subjects, conferring to this epitope a high degree of specificity. This peptide shares also homology with other autoantigens which can be recognized by autoantibodies present in seronegative RA sera. These newly identified autoantibodies, although present also in a percentage of seropositive RA patients, may be considered as novel serum biomarkers for seronegative RA, which lacks the presence of RF and/or ACPAs.

Immunogenic amino acid motifs and linear epitopes of COVID-19 mRNA vaccines

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N = 20 healthy adults, and post-vaccine samples from an additional N = 13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558–569, 627–638, and 1148–1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.

Immunogenic Amino Acid Motifs and Linear Epitopes of COVID-19 mRNA Vaccines

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N=20 healthy adults, and post-vaccine samples from an additional N=13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558-569, 627-638, and 1148-1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.

Phage-display reveals interaction of lipocalin allergen Can f 1 with a peptide resembling the antigen binding region of a human γδT-cell receptor

Although some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction.

Antibody binding epitope Mapping (AbMap) of two hundred antibodies in a single run

Epitope mapping is essential for the understanding how an antibody works. Given millions of antibodies short of epitope information, there is an urgent need for high-throughput epitope mapping. Here we combined a commercial phage displayed random peptide library of 109 diversity with next generation sequencing to develop Antibody binding epitope Mapping (AbMap) technology. Over two hundred antibodies were analyzed in a single test and epitopes were determined for >50% of them. Strikingly, the antibodies were able to recognize different proteins from multiple species with similar epitopes. We successfully identified the epitopes of 14 anti-PD-1 antibodies, including Sintilimab (i.e., L128, A129 and P130), and confirmed that the binding epitopes of Nivolumab and Sintilimab are very close to the binding interface of PD-1 and PD-L1. The predicted conformational epitopes of Pembrolizumab and Nivolumab are consistent with their antibody-antigen co-crystal structures. AbMap is the first technology enables high-throughput epitope mapping.HighlightsThe first technology enables epitope mapping of two hundred antibodies in a single runLinear epitope was determined for >50% of the antibodiesDistinct epitopes of 14 anti-PD-1 antibodies, including Sintilimab, were determinedThe predicted conformational epitopes of Pembrolizumab and Nivolumab are consistent with the known antibody-antigen co-crystal structures