Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5 kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42 kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

Linear epitopes of bony fish ß-parvalbumins.

Introduction: Fish ß-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised. Methods: High-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish ß-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens. Results: Interindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects. Conclusions: Altogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.

Development of a peptide microarray-based metal-enhanced fluorescence assay for ultrasensitive detection of multiple matrix metalloproteinase activities by using a gold nanorod-polymer substrate.

Matrix metalloproteinases (MMPs) are attractive biomarkers for cancer diagnosis and treatment, while it is still a challenge to precise analysis of MMP activities owing to their very low abundance in the biological samples, especially at the early stages of tumors. Herein, a peptide microarray-based metal-enhanced fluorescence assay (PMMEFA) is proposed to simultaneously detect MMP-1, -2, -3, -7, -9, and -13 activities. The assay involves immobilization of Förster resonance energy transfer dye pair decorated peptides (FRET-peptides) on a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) coated gold nanorod modified glass slide (GNR@P(GMA-HEMA)). To fabricate the GNR@P(GMA-HEMA) slide, GNRs are self-assembled onto an aminated glass slide, and a polymer brush (P(GMA-HEMA)) is grown through a surface-initiated atom transfer radical polymerization reaction (SI-ATRP). Upon the addition of MMPs, the FRET pairs are broken due to the specific cleavage of FRET-peptides by enzymes, resulting in the recovery of fluorescence signals and further enhancement by the MEF of GNRs. The fluorescence recovery degree provides a direct indicator for MMP activity. The PMMEFA exhibits excellent sensitivity, which enables to detect MMP-1, -2, -3, -7, -9, and -13 activities, with low limits of detection (LODs) of 1.7 fg mL-1, 0.3 fg mL-1, 2.0 fg mL-1, 1.8 fg mL-1, 2.2 fg mL-1 and 14.0 fg mL-1, respectively. To substantiate the practicability of PMMEFA, MMP activities were measured in a range of matrices, encompassing cell culture medium, serum, and tumor tissue homogenate, and MMP activities can be detected only in 0.15 µL serum and 0.025 mg tumor tissue.

In silico design of a broad-spectrum multiepitope vaccine against influenza virus.

Influenza remains a global health concern due to its potential to cause pandemics as a result of rapidly mutating influenza virus strains. Existing vaccines often struggle to keep up with these rapidly mutating flu viruses. Therefore, the development of a broad-spectrum peptide vaccine that can stimulate an optimal antibody response has emerged as an innovative approach to addressing the influenza threat. In this study, an immunoinformatic approach was employed to rapidly predict immunodominant epitopes from different antigens, aiming to develop an effective multiepitope influenza vaccine (MEV). The immunodominant B-cell linear epitopes of seasonal influenza strains hemagglutinin (HA) and neuraminidase (NA) were predicted using an antibody-peptide microarray, involving a human cohort including vaccinees and infected patients. On the other hand, bioinformatics tools were used to predict immunodominant cytotoxic T-cell (CTL) and helper T-cell (HTL) epitopes. Subsequently, these epitopes were evaluated by various immunoinformatic tools. Epitopes with high antigenicity, high immunogenicity, non-allergenicity, non-toxicity, as well as exemplary conservation were then connected in series with appropriate linkers and adjuvants to construct a broad-spectrum MEV. Moreover, the structural analysis revealed that the MEV candidates exhibited good stability, and the docking results demonstrated their strong affinity to Toll-like receptors 4 (TLR4). In addition, molecular dynamics simulation confirmed the stable interaction between TLR4 and MEVs. Three injections with MEVs showed a high level of B-cell and T-cell immune responses according to the immunological simulations in silico. Furthermore, in-silico cloning was performed, and the results indicated that the MEVs could be produced in considerable quantities in Escherichia coli (E. coli). Based on these findings, it is reasonable to create a broad-spectrum MEV against different subtypes of influenza A and B viruses in silico.

IgM-specific linear epitopes on the E2 protein for serodiagnosis of Chikungunya.

Chikungunya virus (CHIKV) and Dengue virus (DENV) are vector-borne diseases transmitted by Aedes aegypti and Aedes albopictus that pose a significant threat to global public health. Cases of acute Chikungunya fever often present similar clinical symptoms to other vector-borne diseases, such as Dengue fever. In regions where multiple vector-borne diseases coexist, CHIKV is often overlooked or misdiagnosed as Dengue virus, West Nile virus, Zika virus or other viral infections, which delays its prevention and control. However, IgM antibodies directed against the E2 protein of CHIKV have not yet been generalized to clinical settings due to the low sensitivity and high cost in commercial kits. Indirect ELISA with peptides provides an effective supplementary tool for detecting CHIKV IgM antibodies. Our study aims at examining the potential of linear epitopes on the E2 glycoprotein that specifically bind to IgM antibodies as serodiagnostic tool for CHIKV. The sensitivity of the established peptide indirect ELISA method for detecting clinical samples is significantly better than that of commercial kits, realizing a beneficial supplement to the existing IgM antibody assay. It also established the groundwork for comprehending the biological mechanisms of the CHIKV E2 protein and the advancement of innovative epitope peptide vaccines.

Surveillance of SARS-CoV-2 immunogenicity: loss of immunodominant HLA-A*02-restricted epitopes that activate CD8+ T cells.

Introduction and methods: In this present work, coronavirus subfamilies and SARS-CoV-2 Variants of Concern (VOCs) were investigated for the presence of MHC-I immunodominant viral peptides using in silico and in vitro tools. Results: In our results, HLA-A*02 haplotype showed the highest number of immunodominant epitopes but with the lowest combined prediction score. Furthermore, a decrease in combined prediction score was observed for HLA-A*02-restricted epitopes when the original strain was compared to the VOCs, indicating that the mutations on the VOCs are promoting escape from HLA-A2-mediated antigen presentation, which characterizes a immune evasion process. Additionally, epitope signature analysis revealed major immunogenic peptide loss for structural (S) and non-structural (ORF8) proteins of VOCs in comparison to the Wuhan sequence. Discussion: These results may indicate that the antiviral CD8+ T-cell responses generated by original strains could not be sufficient for clearance of variants in either newly or reinfection with SARS-CoV-2. In contrast, N epitopes remain the most conserved and reactive peptides across SARS-CoV-2 VOCs. Overall, our data could contribute to the rational design and development of new vaccinal platforms to induce a broad cellular CD8+ T cell antiviral response, aiming at controlling viral transmission of future SARS-CoV-2 variants.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Synapsin autoantibodies during pregnancy are associated with fetal abnormalities.

Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3% using cell-based assays. Seropositivity was strongly associated with abnormalities of fetal development including structural defects, intrauterine growth retardation, amniotic fluid disorders and neuropsychiatric developmental diseases in previous children (odds ratios of 3-6.5). Autoantibodies reached the fetal circulation and were mainly of IgG1/IgG3 subclasses. They bound to conformational and linear synapsin-I epitopes, five distinct epitopes were identified using peptide microarrays. The findings indicate that synapsin-I autoantibodies may be clinically useful biomarkers or even directly participate in the disease process of neurodevelopmental disorders, thus being potentially amenable to antibody-targeting interventional strategies in the future.

Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting.

Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers.

A linear B-cell epitope close to the furin cleavage site within the S1 domain of SARS-CoV-2 Spike protein discriminates the humoral immune response of nucleic acid- and protein-based vaccine cohorts.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

Characterization and analysis of linear epitopes corresponding to SARS-CoV-2 outbreak in Jilin Province, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants have caused hundreds of thousands of deaths and shown serious social influence worldwide. Jilin Province, China, experienced the first wave of the outbreak from December 2020 to February 2021. Here, we analyzed the genomic characteristics of the SARS-CoV-2 outbreak in Jilin province using a phylogeographic tree and found that clinical isolates belonged to the B.1 lineage, which was considered to be the ancestral lineage. Several dominant SARS-CoV-2 specific linear B cell epitopes that reacted with the convalescent sera were also analysed and identified using a peptide microarray composed of S, M, and E proteins. Moreover, the serum of convalescent patients infected with SARS-CoV-2 showed neutralizing activity against four widely spreading SARS-CoV-2 variants; however, significant differences were observed in neutralizing activities against different SARS-CoV-2 variants. These data provide important information on genomic characteristics, linear epitopes, and neutralizing activity of SARS-CoV-2 outbreak in Jilin Province, China, which may aid in understanding disease patterns and regional aspects of the pandemic.

Applications of Peptide Microarrays in Autoantibody, Infection, and Cancer Detection.

The diversity of the antigen-specific humoral immune response reflects the interaction of the immune system with pathogens and autoantigens. Peptide microarray analysis opens up new perspectives for the use of antibodies as diagnostic biomarkers and provides unique access to a more differentiated view on humoral responses to disease. This review focuses on the latest applications of peptide microarrays for the serologic medical diagnosis of autoimmunity, infectious diseases (including COVID-19), and cancer.

A Bifunctional Polymeric Coating for the Co-Immobilization of Proteins and Peptides on Microarray Substrates.

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules toward a specific target is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule, thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here, we introduce a new N,N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bifunctional copolymer has been obtained by partial post-polymerization modification of the functional groups of a common precursor. This strategy represents a convenient method to reduce the number of analyses, therefore possible systematic or random errors, besides offering a drastic shortage in time, reagents, and costs.

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents.

Profiling of Multiple Matrix Metalloproteinases Activities in the Progression of Osteosarcoma by Peptide Microarray-Based Fluorescence Assay on Polymer Brush-Coated Zinc Oxide Nanorod Substrate.

Peptide microarray provides the ability to miniaturize, parallelize, and automate high-throughput screening substrate specificities of enzymes, profiling of multiple enzyme activities, discovery of disease biomarkers, and development of drugs. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13) activities in the culture medium of four human osteosarcoma (OS) cells and in the progression of OS by using the mouse-bearing xenograft OSs including U-2OS and Saos-2 human. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 with limit of detection downs to 10 pM, 30 pM, 113 pM, 13 pM, 93 pM, and 12 pM, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs is serum closely relevant to the disease progression and type of tumor.

Peptide Microarrays for Flavivirus Diagnosis.

Flavivirus are the most alarming prevalent viruses worldwide due to its vast impact on public health. Most early symptoms of diseases caused by Flavivirus are similar among each other and to other febrile illnesses making the clinical differential diagnosis challenging. In addition, due to cross-reactivity and a relatively limited persistence of viral RNA in infected individuals, the current available diagnosis strategies fail to efficiently provide a differential viral identification. In this context, virus-specific tests are essential to improve patient care, as well as to facilitate disease surveillance and the effective control of transmission. Here, we describe the use of protein microarrays as an effective tool for screening peptides differentially recognized by anti-Yellow Fever virus antibodies induced by vaccination or by natural viral infection.

IgE and IgG4 Epitope Mapping of Food Allergens with a Peptide Microarray Immunoassay.

Peptide microarrays are a powerful tool to identify linear epitopes of food allergens in a high-throughput manner. The main advantages of the microarray-based immunoassay are as follows: the possibility to assay thousands of targets simultaneously, the requirement of a low volume of serum, the more robust statistical analysis, and the possibility to test simultaneously several immunoglobulin subclasses. Among them, the last one has a special interest in the field of food allergy, because the development of tolerance to food allergens has been associated with a decrease in IgE and an increase in IgG4 levels against linear epitopes. However, the main limitation to the clinical use of microarray is the automated analysis of the data. Recent studies mapping the linear epitopes of food allergens with peptide microarray immunoassays have identified peptide biomarkers that can be used for early diagnosis of food allergies and to predict their severity or the self-development of tolerance. Using this approach, we have worked on epitope mapping of the two most important food allergens in the Spanish population, cow's milk, and chicken eggs. The final aim of these studies is to define subsets of peptides that could be used as biomarkers to improve the diagnosis and prognosis of food allergies. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of food allergens and data acquisition and analysis of IgE and IgG4 binding epitopes.

Mapping and Validation of Peptides Differentially Recognized by Antibodies from the Serum of Yellow Fever Virus-Infected or 17DD-Vaccinated Patients.

Yellow Fever disease is caused by the Yellow Fever virus (YFV), an arbovirus from the Flaviviridae family. The re-emergence of Yellow Fever (YF) was facilitated by the increasing urbanization of sylvatic areas, the wide distribution of the mosquito vector, and the low percentage of people immunized in the Americas, which caused severe outbreaks in recent years, with a high mortality rate. Therefore, serological approaches capable of discerning antibodies generated from the wild-type (YFV-WT) strain between the vaccinal strain (YFV-17DD) could facilitate vaccine coverage surveillance, enabling the development of strategies to avoid new outbreaks. In this study, peptides were designed and subjected to microarray procedures with sera collected from individuals infected by WT-YFV and 17DD-YFV of YFV during the Brazilian outbreak of YFV in 2017/2018. From 222 screened peptides, around ten could potentially integrate serological approaches aiming to differentiate vaccinated individuals from naturally infected individuals. Among those peptides, one was synthesized and validated through ELISA.