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

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Mapping the epitopes of Schistosoma japonicum esophageal gland proteins for incorporation into vaccine constructs

Li, Xiao-Hong; Vance, Gillian M.; Cartwright, Jared; Cao, Jian-Ping; Wilson, R Alan; Castro-Borges, William
PLoS ONE.
Feb 2020
10.1371/journal.pone.0229542
Background The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. Methodology/Principal findings We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. Conclusions/Significance It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.

A Protein Epitope Targeted by the Antibody Response to Kawasaki Disease

Rowley, Anne H; Baker, Susan C; Arrollo, David; Gruen, Leah J; Bodnar, Tetyana; Innocentini, Nancy; Hackbart, Matthew; Cruz-Pulido, Yazmin E; Wylie, Kristine M; Kim, Kwang-Youn A; Shulman, Stanford T
Feb 2020
10.1093/infdis/jiaa066
Background Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1–2 weeks after infection. Methods We isolated single peripheral blood plasmablasts from children with KD 1–3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. Results Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients day ≥ 8 after illness onset, compared with 0/17 infant controls (P < .01), recognized the KD peptide antigen. Conclusions These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.

Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer’s disease

Roberts, Malcolm; Sevastou, Ioanna; Imaizumi, Yoichi; Mistry, Kavita; Talma, Sonia; Dey, Madhurima; Gartlon, Jane; Ochiai, Hiroshi; Zhou, Zhi; Akasofu, Shigeru; Tokuhara, Naoki; Ogo, Makoto; Aoyama, Muneo; Aoyagi, Hirofumi; Strand, Kate; Sajedi, Ezat; Agarwala, Kishan Lal; Spidel, Jared; Albone, Earl; Horie, Kanta; Staddon, James M.; de Silva, Rohan
Acta Neuropathologica Communications.
Feb 2020
10.1186/s40478-020-0884-2
Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (“seeds”) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Immunization with full-length Plasmodium falciparum merozoite surface protein 1 is safe and elicits functional cytophilic antibodies in a randomized first-in-human trial

Blank, Antje; Fürle, Kristin; Jäschke, Anja; Mikus, Gerd; Lehmann, Monika; Hüsing, Johannes; Heiss, Kirsten; Giese, Thomas; Carter, Darrick; Böhnlein, Ernst; Lanzer, Michael; Haefeli, Walter E.; Bujard, Hermann
npj Vaccines.
Jan 2020
10.1038/s41541-020-0160-2
A vaccine remains a priority in the global fight against malaria. Here, we report on a single-center, randomized, double-blind, placebo and adjuvant-controlled, dose escalation phase 1a safety and immunogenicity clinical trial of full-length Plasmodium falciparum merozoite surface protein 1 (MSP1) in combination with GLA-SE adjuvant. Thirty-two healthy volunteers were vaccinated at least three times with MSP1 plus adjuvant, adjuvant alone, or placebo (24:4:4) to evaluate the safety and immunogenicity. MSP1 was safe, well tolerated and immunogenic, with all vaccinees sero-converting independent of the dose. The MSP1-specific IgG and IgM titers persisted above levels found in malaria semi-immune humans for at least 6 months after the last immunization. The antibodies were variant- and strain-transcending and stimulated respiratory activity in granulocytes. Furthermore, full-length MSP1 induced memory T-cells. Our findings encourage challenge studies as the next step to evaluate the efficacy of full-length MSP1 as a vaccine candidate against falciparum malaria (EudraCT 2016-002463-33).

The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context

Schüchner, Stefan; Behm, Christian; Mudrak, Ingrid; Ogris, Egon
Sci. Signal..
Jan 2020
10.1126/scisignal.aax9730
Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.

Serum levels of sclerostin reflect altered bone microarchitecture in patients with hepatic cirrhosis

Wakolbinger, Robert; Muschitz, Christian; Wallwitz, Jacqueline; Bodlaj, Gerd; Feichtinger, Xaver; Schanda, Jakob E.; Resch, Heinrich; Baierl, Andreas; Pietschmann, Peter
Wien Klin Wochenschr.
Jan 2020
10.1007/s00508-019-01595-8
Background: Patients with hepatic cirrhosis are at increased risk of bone loss. Recent work on areal bone mineral density has reported contradictory findings. As the assessment of bone microarchitecture is complex, a search was made for correlations with new serum markers of bone turnover. Current data on serum sclerostin levels in patients with increased fracture risk are divergent and to date only one study has examined patients with hepatic cirrhosis. Therefore, the aim of this study was to evaluate serum sclerostin levels and to test for correlations with microarchitecture. Methods: This study was performed in 32 patients with recently diagnosed hepatic cirrhosis and 32 controls. The parameters of bone microarchitecture were assessed by high-resolution peripheral quantitative computed tomography. Sclerostin was detected via a new ELISA that detects the active receptor interaction site at loop 2 of the sclerostin core region. Results: Sclerostin levels were slightly, but not significantly lower in the patient group, compared to controls. In contrast, patients with alcoholic liver cirrhosis had significantly lower levels than the controls. A significant correlation with areal bone mineral density (BMD) and trabecular microarchitecture was observed in the patient group. However, there was hardly any correlation between sclerostin and bone microarchitecture in the controls. Conclusion: In hepatic cirrhosis, sclerostin is related to altered bone microarchitecture and lower areal BMD. In alcoholic liver disease, low sclerostin concentrations were seen.

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

Dreyfus, David H.; Farina, Antonella; Farina, Giuseppina Alessandra
Immunol Res.
Dec 2018
10.1007/s12026-018-9045-0
EBV (Epstein-Barr Virus) and other human DNA viruses are associated with autoimmune syndromes in epidemiologic studies. In this work, immunoglobulin G response to EBV-encoded proteins which share regions with human immune response proteins from the human host including ZEBRA (BZLF-1 encoded protein), BALF-2 recombinase expressed primarily during the viral lytic replication cycle, and EBNA-1 (Epstein-Barr Virus Nuclear Antigen) expressed during the viral latency cycle respectively were characterized using a laser-printed micro-array (PEPperprint.com). IgG response to conserved “A/T hooks” in EBV-encoded proteins such as EBNA-1 and the BALF-2 recombinase related to host DNA-binding proteins including RAG-1 recombinase and histones, and EBV-encoded virokines such as the IL-10 homologue BCRF-1 suggest further directions for clinical research. The author suggests that proteomic “molecular fingerprints” of the immune response to viral proteins shared with human immune response genes are potentially useful in early diagnosis and monitoring of autoantibody production and response to therapy in EBV-related autoimmune syndromes.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease

Haeberle, Stefanie; Wei, Xiaoying; Bieber, Katja; Goletz, Stephanie; Ludwig, Ralf J.; Schmidt, Enno; Enk, Alexander H.; Hadaschik, Eva N.
Journal of Allergy and Clinical Immunology.
Dec 2018
10.1016/j.jaci.2018.04.006
Background Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. Objective We sought to determine whether AIBDs develop in Treg cell–deficient scurfy mice. Methods Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Results Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell–deficient nu/nu mice. Conclusion We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Soybean Allergy Related Epitopes

Kern, Karolin; Spiegel, Holger; Havenith, Heide; Szardenings, Michael
Nov 2018
The invention relates to a compilation comprising at least five different peptides, each peptide comprising at least one sequence element corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. The invention further relates to an in vitro method for determining a patient’s immune status to soybean allergens, to a method for detecting at least one soybean allergen in a substance and to a method for determining the allergenicity of a soybean variety. Additionally, the invention relates to a kit comprising at least one composition containing a compound comprising at least five different sequence elements each corresponding to an epitope selected from the group consisting of SEQ ID NO.: 1-354, wherein at least five different epitopes are represented. Furthermore, the invention relates to the use of a peptide comprising a sequence element corresponding to an epitope for providing a molecule binding to a protein or peptide comprising the epitope.

Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins

Lagatie, Ole; Verheyen, Ann; Van Dorst, Bieke; Batsa Debrah, Linda; Debrah, Alex; Stuyver, Lieven J.
Parasite Immunol.
Nov 2018
10.1111/pim.12587
In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2, Ov-ALT-1, Ov-RAL-2, Ov-ASP-1, Ov-103, Ov-RBP-1, Ov-CHI-1, and Ov-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.

Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

Lee, Yeon-Ah; Hahm, Dae-Hyun; Kim, Jung Yeon; Sur, Bonjun; Lee, Hyun Min; Ryu, Chun Jeih; Yang, Hyung-In; Kim, Kyoung Soo
Arthritis Res Ther.
Oct 2018
10.1186/s13075-018-1736-3
Background Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. Methods Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. Results The mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. Conclusions mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.

Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin

Aliprandini, Eduardo; Tavares, Joana; Panatieri, Raquel Hoffmann; Thiberge, Sabine; Yamamoto, Marcio Massao; Silvie, Olivier; Ishino, Tomoko; Yuda, Masao; Dartevelle, Sylvie; Traincard, François; Boscardin, Silvia Beatriz; Amino, Rogerio
Nat Microbiol.
Oct 2018
10.1038/s41564-018-0254-z
The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic ‘dotty death’ phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving, or migrate and traverse cells to progress through the host tissues at the eventual expense of their own life.

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