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

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Peptide microarray IgM and IgG screening of pre-SARS-CoV-2 human serum samples from Zimbabwe for reactivity with peptides from all seven human coronaviruses: a cross-sectional study

Ashworth, Jordan; Mathie, Dayna; Scott, Fiona; Mahendran, Yuvaraj; Woolhouse, Mark; Stoevesandt, Oda; Mduluza, Takafira; Mutapi, Francisca
The Lancet Microbe.
Feb 2023
10.1016/S2666-5247(22)00295-6

Anti-brolucizumab immune response as one prerequisite for rare retinal vasculitis/retinal vascular occlusion adverse events

Karle, Anette C.; Wrobel, Matthias B.; Koepke, Stephan; Gutknecht, Michael; Gottlieb, Sascha; Christen, Brigitte; Rubic-Schneider, Tina; Pruimboom-Brees, Ingrid; Leber, Xavier Charles; Scharenberg, Meike; Maciejewski, Benjamin; Turner, Oliver; Saravanan, Chandra; Huet, Francois; Littlewood-Evans, Amanda; Clemens, Andreas; Grosskreutz, Cynthia L.; Kearns, Jeffrey D.; Mehan, Pawan; Schmouder, Robert L.; Sasseville, Vito; Brees, Dominique
Sci. Transl. Med..
Feb 2023
10.1126/scitranslmed.abq5241
In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.

Human antibody profiling technologies for autoimmune disease

Carlton, Lauren H.; McGregor, Reuben; Moreland, Nicole J.
Immunol Res.
Jan 2023
10.1007/s12026-023-09362-8
Abstract Autoimmune diseases are caused by the break-down in self-tolerance mechanisms and can result in the generation of autoantibodies specific to human antigens. Human autoantigen profiling technologies such as solid surface arrays and display technologies are powerful high-throughput technologies utilised to discover and map novel autoantigens associated with disease. This review compares human autoantigen profiling technologies including the application of these approaches in chronic and post-infectious autoimmune disease. Each technology has advantages and limitations that should be considered when designing new projects to profile autoantibodies. Recent studies that have utilised these technologies across a range of diseases have highlighted marked heterogeneity in autoantibody specificity between individuals as a frequent feature. This individual heterogeneity suggests that epitope spreading maybe an important mechanism in the pathogenesis of autoimmune disease in general and likely contributes to inflammatory tissue damage and symptoms. Studies focused on identifying autoantibody biomarkers for diagnosis should use targeted data analysis to identify the rarer public epitopes and antigens, common between individuals. Thus, utilisation of human autoantigen profiling technology, combined with different analysis approaches, can illuminate both pathogenesis and biomarker discovery.

Antibody isotype epitope mapping of SARS‐CoV‐2 Spike RBD protein: targets for COVID‐19 symptomatology and disease control

Contreras, Marinela; Vicente, Joaquín; Cerón, José Joaquín; Subiela, Silvia Martinez; Urra, José Miguel; Rodríguez‐del‐Río, Francisco J.; Ferreras‐Colino, Elisa; Vaz‐Rodrigues, Rita; de Mera, Isabel G. Fernández; Antunes, Sandra; Domingos, Ana; Gortázar, Christian; de la Fuente, José
Eur J Immunol.
Jan 2023
10.1002/eji.202250206

Antibody Properties Associate with Clinical Phenotype in LGI1 Encephalitis

Ludewig, Susann; Salzburger, Leonie; Goihl, Alexander; Rohne, Jana; Leypoldt, Frank; Bittner, Daniel; Düzel, Emrah; Schraven, Burkhart; Reinhold, Dirk; Korte, Martin; Körtvélyessy, Péter
Cells.
Jan 2023
10.3390/cells12020282
Autoimmune encephalitis (AE) associated with autoantibodies against leucine-rich glioma-inactivated protein-1 (LGI1) can present with faciobrachial dystonic seizures (FBDS) and/or limbic encephalitis (LE). The reasons for this heterogeneity in phenotypes are unclear. We performed autoantibody (abs) characterization per patient, two patients suffering from LE and two from FBDS, using isolated antibodies specified with single amino acid epitope mapping. Electrophysiological slice recordings were conducted alongside spine density measurements, postsynaptic Alpha-amino-3-hydoxy-5-methyl-4-isoaxole-proprionate-receptors (AMPA-R) and N-methyl-D-aspartate-receptors receptor (NMDA-R) cluster counting. These results were correlated with the symptoms of each patient. While LGI1 abs from LE patients mainly interacted with the Leucine-rich repeat section of LGI1, abs from both FBDS patients also recognized the Epitempin section as well. Six-hour incubation of mouse hippocampal slices with LE patients autoantibodies but not from the FBDS patients resulted in a significant decline in long-term potentiation (p = 0.0015) or short-term plasticity at CA3-CA1 neurons and in decreased hippocampal synaptic density. Cluster differentiation showed no decrease in postsynaptic AMPA-R and NMDA-R. LGI1 autoantibodies selected by phenotype show an almost distinct epitope pattern, elicit disparate functional effects on hippocampal neurons, and cause divergent effects on spine density. This data illuminates potential pathomechanisms for disease heterogeneity in LGI1 AE.

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.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples

He, Xiaohua; Patfield, Stephanie; Hnasko, Robert; Rasooly, Reuven; Mandrell, Robert E.
PLoS ONE.
Oct 2013
10.1371/journal.pone.0076368
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Provocation of an Autoimmune Response to Cardiac Voltage-Gated Sodium Channel NaV1.5 Induces Cardiac Conduction Defects in Rats

Korkmaz, Sevil; Zitron, Edgar; Bangert, Anna; Seyler, Claudia; Li, Shiliang; Hegedüs, Peter; Scherer, Daniel; Li, Jin; Fink, Thomas; Schweizer, Patrick A.; Giannitsis, Evangelos; Karck, Matthias; Szabó, Gábor; Katus, Hugo A.; Kaya, Ziya
Journal of the American College of Cardiology.
Jul 2013
10.1016/j.jacc.2013.04.041
Objectives: This study sought to test the hypothesis that inducing an autoimmune response against the cardiac sodium channel (NaV1.5) induces arrhythmias. Background: Sporadic evidence supports the concept that autoantibodies may cause cardiac arrhythmias but substantial experimental investigations using in vivo models have been lacking to date. The NaV1.5 is essential for cardiac impulse propagation and its dysfunction has been linked to conduction disease. Methods: Rats were immunized with a peptide sequence derived from the third extracellular loop of the first domain of NaV1.5. After 28 days, we evaluated in vivo both the electrical and mechanical parameters of cardiac function. Histopathology, myocardial gene and protein expression were assessed. Whole-cell patch-clamp was used to measure sodium current (INa) density in isolated cardiomyocytes. Results: NaV1.5-immunized rats had high titers of autoantibodies against NaV1.5. On ECG recording, NaV1.5-immunized animals showed significantly prolonged PR-intervals. During Holter ECG-monitoring we observed repeated prolonged episodes of third-degree atrioventricular and sinoatrial block in every NaV1.5-immunized animal, but not in controls. Immunization had no effect on cardiac function. In comparison to controls, myocardial NaV1.5 mRNA and protein levels were decreased in immunized rats. INa density was reduced in cardiomyocytes incubated with sera from NaV1.5-immunized rats and from patients with idiopathic atrioventricular block (AVB) in comparison to sera from respective controls. In patients with idiopathic AVB, we observed autoantibodies against NaV1.5 that were absent in sera from healthy controls. Conclusions: Provocation of an autoimmune response against NaV1.5 induces conductance defects probably caused by a reduced expression level and an inhibition of NaV1.5 by autoantibodies, resulting in decreased INa.

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

Heilkenbrinker, Uta; Dietrich, Richard; Didier, Andrea; Zhu, Kui; Lindbäck, Toril; Granum, Per Einar; Märtlbauer, Erwin
PLoS ONE.
Apr 2013
10.1371/journal.pone.0063104
The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml−1, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Shukla, Nikunj M.; Salunke, Deepak B.; Balakrishna, Rajalakshmi; Mutz, Cole A.; Malladi, Subbalakshmi S.; David, Sunil A.
PLoS ONE.
Aug 2012
10.1371/journal.pone.0043612
Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreacti

Sensing Immune Responses with Customized Peptide Microarrays

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Biointerphases.
Aug 2012
10.1007/s13758-012-0047-5
The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

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