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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

Oliveira, Jamille Ramos; Ruiz, Cesar Manuel Remuzgo; Machado, Rafael Rahal Guaragna; Magawa, Jhosiene Yukari; Daher, Isabela Pazotti; Urbanski, Alysson Henrique; Schmitz, Gabriela Justamante Händel; Arcuri, Helen Andrade; Ferreira, Marcelo Alves; Sasahara, Greyce Luri; de Medeiros, Giuliana Xavier; Júnior, Roberto Carlos Vieira Silva; Durigon, Edison Luiz; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro; Schechtman, Deborah; Nakaya, Helder I.; Cunha-Neto, Edecio; Gadermaier, Gabriele; Kalil, Jorge; Coelho, Verônica; Santos, Keity Souza
Front. Immunol..
Jan 2023
10.3389/fimmu.2022.1010105
Introduction Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information. Methods We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals. Results We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity. Discussion This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Deciphering the Autoantibody Response to the OJ Antigenic Complex

Fritzler, Marvin J.; Bentow, Chelsea; Satoh, Minoru; McHugh, Neil; Ghirardello, Anna; Mahler, Michael
Diagnostics.
Jan 2023
10.3390/diagnostics13010156
(1) Background: Myositis specific antibodies (MSA) are important diagnostic biomarkers. Among the rarest and most challenging MSA are anti-OJ antibodies which are associated with anti-synthetase syndrome (ASS). In contrast to the other tRNA synthetases that are targets of ASS autoantibodies (e.g Jo-1, PL-7, PL-12, EJ, KS, Zo), OJ represents a macromolecular complex with several ribonucleoprotein subunits. Therefore, the choice of the antigen in autoantibody assays can be challenging. (2) Methods: We collected two independent cohorts with anti-OJ antibodies, one based on a commercial line immunoassay (LIA) (n = 39), the second based on protein immunoprecipitation (IP) (n = 15). Samples were tested using a particle-based multi-analyte technology (PMAT) system that allows for the simultaneous detection of antibodies to various autoantigens. For the detection of anti-OJ antibodies, two different antigens were deployed (KARS, IARS) on PMAT. The reactivity to the two antigens KARS and IARS was analyzed individually and combined in a score (sum of the median fluorescence intensities). (3) Results: In the cohort selection based on LIA, 3/39 (7.7%) samples were positive for anti-KARS and 7/39 (17.9%) for anti-IARS and 14/39 (35.9%) when the two antigens were combined. In contrast, in samples selected by IP the sensitivity of anti-KARS was higher: 6/15 (40.0%) samples were positive for anti-KARS, 4/15 (26.7%) for anti-IARS and 12/15 (80.0%) for the combination of the two antigens. 18/39 (46.2%) of the LIA samples generated a cytoplasmic IIF pattern (compatible with anti-synthetase antibodies), but there was no association with the antibody levels, neither with LIA nor with PMAT. (4) Conclusions: The combination of IARS and KARS might represent a promising approach for the detection of anti-OJ antibodies on a fully automated platform.

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains

Luckey, David; Weaver, Eric A.; Osborne, Douglas G.; Billadeau, Daniel D.; Taneja, Veena
Sci Rep.
Dec 2019
10.1038/s41598-019-55503-1
Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

Analysis of humoral immune responses in chikungunya virus (CHIKV) infected patients and individuals vaccinated with a candidate CHIKV vaccine

Henss, Lisa; Yue, Constanze; von Rhein, Christine; Tschismarov, Roland; Lewis-Ximenez, Lia Laura; Dölle, Albert; Baylis, Sally A; Schnierle, Barbara S
Dec 2019
10.1093/infdis/jiz658
Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. Results Greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2–6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. Conclusion These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Peptides of neuron specific enolase as potential ASD biomarkers: From discovery to epitope mapping

Ramirez-Celis, Alexandra; Edmiston, Elizabeth; Schauer, Joseph; Vu, Tam; Van de Water, Judy
Brain, Behavior, and Immunity.
Dec 2019
10.1016/j.bbi.2019.12.002
Autism spectrum disorder (ASD) is an important health issue and affects 1 in 59 children in the US. Prior studies determined that maternal autoantibody related (MAR) autism is thought to be associated with ~23% of ASD cases. We previously identified seven MAR-specific autoantigens including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1. We subsequently described the epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens. The aim of the current study was to expand upon our previous work and identify additional antigens recognized by the ASD-specific maternal autoantibodies, as well as to map the unique ASD-specific epitopes using microarray technology. Fetal Rhesus macaque brain tissues were separated by molecular weight and a fraction containing bands between 37 and 45 kDa was analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF/TOF tandem MS/MS. Using this methodology, Neuron specific enolase (NSE) was identified as a target autoantigen and selected for epitope mapping. The full NSE sequence was translated into 15-mer peptides with an overlap of 14 amino acids onto microarray slides and probed with maternal plasma from mothers with an ASD child and from mothers with a Typically Developing child (TD) (ASD = 27 and TD = 21). The resulting data were analyzed by T-test. We found 16 ASD-specific NSE-peptide sequences for which four sequences were statistically significant (p < 0.05) using both the t-test and SAM t-test: DVAASEFYRDGKYDL (p = 0.047; SAM score 1.49), IEDPFDQDDWAAWSK (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (p = 0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (p = 0.017; SAM score 1.82). We further identified 5 sequences that were recognized by both ASD and TD antibodies suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW). While maternal autoantibodies against the NSE protein are present both in mothers with ASD and mothers of TD children, there are several ASD-specific epitopes that can potentially be used as MAR ASD biomarkers. Further, studies including analysis of NSE as a target protein in combination with the previously identified MAR ASD autoantigens are currently underway.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays

Eickelmann, Stephan; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Zhang, Junfang; Molinari, Valerio; Mende, Marco; Loeffler, Felix F.
Adv. Mater. Technol..
Nov 2019
10.1002/admt.201900503
A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

Ara h 7 isoforms share many linear epitopes: Are 3D epitopes crucial to elucidate divergent abilities?

Ehlers, Anna M.; Klinge, Marco; Suer, Waltraud; Weimann, Yvonne; Knulst, André C.; Besa, Frithjof; Le, Thuy‐My; Otten, Henny G.
Clin Exp Allergy.
Nov 2019
10.1111/cea.13496
Background The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature. Objective To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation. Methods Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15-mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20-mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls (validation). Results Three out of 14 linear epitopes were unique for each isoform (Ara h 7.0101: aa 97-109; Ara h 7.0201: aa 122-133; Ara h 7.0301: aa 65-74) but scarcely recognized by IgE. The main linear IgE epitope (aa 51-57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients (discovery and validation cohort). Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55-65), whereas epitope aa 129-137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z-score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE. Conclusion & Clinical Relevance Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes.

Immunization of mice with chimeric antigens displaying selected epitopes confers protection against intestinal colonization and renal damage caused by Shiga toxin-producing Escherichia coli

Montero, David A.; Del Canto, Felipe; Salazar, Juan C.; Cespedes, Sandra; Cádiz, Leandro; Arenas-Salinas, Mauricio; Reyes, José; Oñate, Ángel; Vidal, Roberto M.
Sep 2019
10.1101/783829
Abstract Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7 and the intranasal conferred protection against renal damage caused by STEC O91:H21. This pre-clinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin‐1

Gadermaier, Elisabeth; Tesarz, Manfred; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
J Clin Lab Anal.
Sep 2019
10.1002/jcla.22944
Background Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. Methods We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. Results The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. Conclusion Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B

Fritzler, Marvin J.; Brown, Rachael D.; Zhang, Meifeng
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.
Aug 2019
10.1089/mab.2019.0016
Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

Genomics-Driven Immunoproteomics: An Integrative Platform to Uncover Important Biomarkers for Human Diseases

Giri, Raghavendra; Qendro, Veneta; Rani, Pooja; Jepchumba, Carren; Bugos, Grace; Stadler, Volker; Han, David K.
Jul 2019
10.1007/978-1-4939-9597-4_21
Genomics-driven immunoproteomics (GDI) is a platform that helps identify antigenic protein targets of mutations and other deoxyribonucleic acid (DNA) variations that are commonly associated with pathological states. This platform utilizes data generated from deep sequencing of exomic DNA or ribonucleic acid (RNA) as input to synthesize mutant peptides into microarrays, which then can be used to detect antigenic proteins that invoke immune response in patients. The technology has been used to detect antigenic targets of multiple sclerosis, an autoimmune disease [1], and cancer to identify mutant proteins that invoke immune response in breast cancer patients [2]. This technology has many potential applications to select genomic changes that are specifically recognized by the immune system in a rapid and efficient manner.

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