Mimotopes for Mycotoxins Diagnosis Based on Random Peptides or Recombinant Antibodies from Phage Library

Mycotoxins, the small size secondary metabolites of fungi, have posed a threat to the safety of medicine, food and public health. Therefore, it is essential to create sensitive and effective determination of mycotoxins. Based on the special affinity between antibody and antigen, immunoassay has been proved to be a powerful technology for the detection of small analytes. However, the tedious preparation and instability of conventional antibodies restrict its application on easy and fast mycotoxins detection. By virtue of simplicity, ease of use, and lower cost, phage display library provides novel choices for antibodies or hapten conjugates, and lead random peptide or recombinant antibody to becoming the promising and environmental friendly immune-reagents in the next generation of immunoassays. This review briefly describes the latest developments on mycotoxins detection using M13 phage display, mainly focusing on the recent applications of phage display technology employed in mycotoxins detection, including the introduction of phage and phage display, the types of phage displayed peptide/recombinant antibody library, random peptides/recombinant antibodies-based immunoassays, as well as simultaneous determination of multiple mycotoxins.

Factor VIII Epitope Analysis Using a Random Peptide Phage-Display Library Approach in the Sippet Cohort

Background Inhibitor development is the most severe complication of hemophilia A care, and is associated with increased morbidity and mortality. Aims The aim of this study was to use a novel epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population. Methods The population consisted of 122 previously untreated patients with severe hemophilia A that were followed-up for 50 days of exposure to FVIII. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII-specific IgG epitope repertoire was assessed by means of a novel high-throughput epitope mapping technique using a random peptide phage-display library. Using this assay, a set of affinity-selected 12-mer peptide sequences (also called mimotopes) that were strongly bound by FVIII-specific antibodies were identified. These mimotopes were clustered on the basis of sequence similarity and a consensus motif was generated for each mimotope cluster. Discriminative performance of these mimotope clusters was assessed by ROC analysis. Mimotope clusters were mapped onto the 3D structure of a B-domain deleted FVIII model using a B-cell epitope prediction algorithm (Mapitope). Results The FVIII-specific antibody response is polyclonal with several mimotope clusters. The most predominant mimotope clusters in inhibitor patients were mapped to the heavy chain of the FVIII molecule. Using plasma samples taken before exposure to FVIII, three mimotopes (with the consensus motifs "QM", "PSLxWK" and "SWPHxxxxK") were identified that predicted inhibitor development (with an AUC of 0.76, 0.80 and 0.76 respectively). Conclusion Information on immunodominant epitope clusters can be used to generate novel, less immunogenic FVIII proteins and set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII. Figure 1 Figure 1. Disclosures Palm: Protobios LLC: Current Employment, Patents & Royalties: Inventor of the patent application (PCT Application No. US/14079626) filed by Protobios that covers the use of phage display method to manipulate and monitor humoral immunity.. Palla: Pfizer: Other: Travel support; Kedrion: Other: Travel support; Novonordisk: Speakers Bureau. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

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.

Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.

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.

Scoping review of the applications of peptide microarrays on the fight against human infections.

Introduction This scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis. Methods Electronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines. Results Eighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010–2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling. Conclusion The most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

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.

Predicting mucin-type O-glycosylation using enhancement value products from derived protein features

Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins. This glycosylation is initiated in the Golgi by the addition of the sugar N-acetylgalactosamine (GalNAc) onto protein Ser and Thr residues by a family of polypeptide GalNAc transferases. In humans, there are 20 isoforms that are differentially expressed across tissues that serve multiple important biological roles. Using random peptide substrates, isoform specific amino acid preferences have been obtained in the form of enhancement values (EV). These EVs alone have previously been used to predict O-glycosylation sites via the web based ISOGlyP (Isoform Specific O-Glycosylation Prediction) tool. Here, we explore additional protein features to determine whether these can complement the random peptide derived enhancement values and increase the predictive power of ISOGlyP. The inclusion of additional protein substrate features (such as secondary structure and surface accessibility) was found to increase sensitivity with minimal loss of specificity, when tested with three different published in vivo O-glycoproteomics data sets, thus increasing the overall accuracy of the ISOGlyP predictions.