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

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Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi

Versteeg, Leroy; Adhikari, Rakesh; Poveda, Cristina; Villar-Mondragon, Maria Jose; Jones, Kathryn M.; Hotez, Peter J.; Bottazzi, Maria Elena; Tijhaar, Edwin; Pollet, Jeroen
PLoS Negl Trop Dis.
Sep 2021
10.1371/journal.pntd.0009689
Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Lee, Hye Lim; Park, Jin-Woo; Seok, Jin Myoung; Jeon, Mi Young; Kim, Hojin; Lim, Young-Min; Shin, Ha Young; Kang, Sa-Yoon; Kwon, Oh-Hyun; Lee, Sang-Soo; Seok, Hung Youl; Min, Ju-Hong; Lee, Sung-Hyun; Kim, Byung-Jo; Kim, Byoung Joon
Diagnostics.
Jul 2021
10.3390/diagnostics11081339
Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Identification of a Zika NS2B epitope as a biomarker for severe clinical phenotypes

Loeffler, Felix F.; Viana, Isabelle F. T.; Fischer, Nico; Coêlho, Danilo F.; Silva, Carolina S.; Purificação, Antônio F.; Araújo, Catarina M. C. S.; Leite, Bruno H. S.; Durães-Carvalho, Ricardo; Magalhães, Tereza; Morais, Clarice N. L.; Cordeiro, Marli T.; Lins, Roberto D.; Marques, Ernesto T. A.; Jaenisch, Thomas
RSC Med. Chem..
Jul 2021
10.1039/D1MD00124H
The identification of specific biomarkers for Zika infection and its clinical complications is fundamental to mitigate the infection spread, which has been associated with a broad range of neurological sequelae. , The identification of specific biomarkers for Zika infection and its clinical complications is fundamental to mitigate the infection spread, which has been associated with a broad range of neurological sequelae. We present the characterization of antibody responses in serum samples from individuals infected with Zika, presenting non-severe (classical) and severe (neurological disease) phenotypes, with high-density peptide arrays comprising the Zika NS1 and NS2B proteins. The data pinpoints one strongly IgG-targeted NS2B epitope in non-severe infections, which is absent in Zika patients, where infection progressed to the severe phenotype. This differential IgG profile between the studied groups was confirmed by multivariate data analysis. Molecular dynamics simulations and circular dichroism have shown that the peptide in solution presents itself in a sub-optimal conformation for antibody recognition, which led us to computationally engineer an artificial protein able to stabilize the NS2B epitope structure. The engineered protein was used to interrogate paired samples from mothers and their babies presenting Zika-associated microcephaly and confirmed the absence of NS2B IgG response in those samples. These findings suggest that the assessment of antibody responses to the herein identified NS2B epitope is a strong candidate biomarker for the diagnosis and prognosis of Zika-associated neurological disease.

Landscape and selection of vaccine epitopes in SARS-CoV-2

Smith, Christof C.; Olsen, Kelly S.; Gentry, Kaylee M.; Sambade, Maria; Beck, Wolfgang; Garness, Jason; Entwistle, Sarah; Willis, Caryn; Vensko, Steven; Woods, Allison; Fini, Misha; Carpenter, Brandon; Routh, Eric; Kodysh, Julia; O’Donnell, Timothy; Haber, Carsten; Heiss, Kirsten; Stadler, Volker; Garrison, Erik; Sandor, Adam M.; Ting, Jenny P. Y.; Weiss, Jared; Krajewski, Krzysztof; Grant, Oliver C.; Woods, Robert J.; Heise, Mark; Vincent, Benjamin G.; Rubinsteyn, Alex
Genome Medicine.
Jun 2021
10.1186/s13073-021-00910-1
Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).

Non-invasive immunoPET imaging of PD-L1 using anti-PD-L1-B11 in breast cancer and melanoma tumor model

Bansal, Aditya; Pandey, Mukesh K.; Barham, Whitney; Liu, Xin; Harrington, Susan M.; Lucien, Fabrice; Dong, Haidong; Park, Sean S.; DeGrado, Timothy R.
Nuclear Medicine and Biology.
May 2021
10.1016/j.nucmedbio.2021.05.004
Introduction Immunotherapy targeting PD-1/PD-L1 immune checkpoint inhibition (ICI) is efficacious in various solid and hematologic malignancies. However, the response rate to PD-1/PD-L1 therapy is only 15–35%. To obtain optimal clinical response to ICI therapies, a reliable assessment of tumor PD-L1 expression is needed to select appropriate patients, and a non-invasive imaging-based assessment of PD-L1 expression is critically needed. Although radiolabeled PET probes based on PD-L1 targeted therapeutic antibodies (e.g. atezolizumab) have shown encouraging results, there is concern that residual therapeutic antibody may compete for binding with the radiotracer thereby compromising imaging studies that follow treatment. Methods and results In this study, we used novel anti-PD-L1-B11 clone antibody known to bind to a different epitope of PD-L1 than the therapeutic antibodies to avoid potential saturation effects. The anti-PD-L1-B11 clone was radiolabeled with zirconium-89 and evaluated to detect PD-L1 expression in various in vitro and in vivo cancer model systems in comparison with [89Zr]Zr-DFO-NCS-atezolizumab. In vitro binding parameters of anti-PD-L1-B11 were like those of atezolizumab. [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 clone showed favorable properties to [89Zr]Zr-DFO-NCS-atezolizumab in an in vivo breast cancer tumor model system with higher uptake in PD-L1 expressing tumors. Conclusion Our data demonstrates that [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 exhibits excellent imaging properties for the assessment PD-L1 expression. The independent binding site of anti-PD-L1-B11 relative to therapeutic anti-PD-L1 antibodies may be advantageous for anti-PD-L1 therapy monitoring.

SARS-CoV-2 spike protein stabilized in the closed state induces potent neutralizing responses.

Carnell, George W.; Ciazynska, Katarzyna A.; Wells, David A.; Xiong, Xiaoli; Aguinam, Ernest T.; McLaughlin, Stephen H.; Mallery, Donna; Ebrahimi, Soraya; Ceron-Gutierrez, Lourdes; Asbach, Benedikt; Einhauser, Sebastian; Wagner, Ralf; James, Leo C.; Doffinger, Rainer; Heeney, Jonathan L.; Briggs, John A. G.
May 2021
10.1128/JVI.00203-21
The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern B.1.248 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines. Importance Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next generation vaccines.

Longitudinal Development of Antibody Responses in COVID-19 Patients of Different Severity with ELISA, Peptide, and Glycan Arrays: An Immunological Case Series

Heidepriem, Jasmin; Dahlke, Christine; Kobbe, Robin; Santer, René; Koch, Till; Fathi, Anahita; Seco, Bruna M. S.; Ly, My L.; Schmiedel, Stefan; Schwinge, Dorothee; Serna, Sonia; Sellrie, Katrin; Reichardt, Niels-Christian; Seeberger, Peter H.; Addo, Marylyn M.; Loeffler, Felix F.; on behalf of the ID-UKE COVID-19 Study Group
Apr 2021
10.3390/pathogens10040438
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). A better understanding of its immunogenicity can be important for the development of improved diagnostics, therapeutics, and vaccines. Here, we report the longitudinal analysis of three COVID-19 patients with moderate (#1) and mild disease (#2 and #3). Antibody serum responses were analyzed using spike glycoprotein enzyme linked immunosorbent assay (ELISA), full-proteome peptide, and glycan microarrays. ELISA immunoglobulin A, G, and M (IgA, IgG, and IgM) signals increased over time for individuals #1 and #2, whereas #3 only showed no clear positive IgG and IgM result. In contrast, peptide microarrays showed increasing IgA/G signal intensity and epitope spread only in the moderate patient #1 over time, whereas early but transient IgA and stable IgG responses were observed in the two mild cases #2 and #3. Glycan arrays showed an interaction of antibodies to fragments of high-mannose and core N-glycans, present on the viral shield. In contrast to protein ELISA, microarrays allow for a deeper understanding of IgA, IgG, and IgM antibody responses to specific epitopes of the whole proteome and glycans of SARS-CoV-2 in parallel. In the future, this may help to better understand and to monitor vaccination programs and monoclonal antibodies as therapeutics.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Bund, Timo; Nikitina, Ekaterina; Chakraborty, Deblina; Ernst, Claudia; Gunst, Karin; Boneva, Boyana; Tessmer, Claudia; Volk, Nadine; Brobeil, Alexander; Weber, Achim; Heikenwalder, Mathias; Zur Hausen, Harald; de Villiers, Ethel-Michele
Proc Natl Acad Sci U S A.
Mar 2021
10.1073/pnas.2025830118
Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

SARS-CoV-2 proteome-wide analysis revealed significant epitope signatures in COVID-19 patients

Schwarz, Tatjana; Heiss, Kirsten; Mahendran, Yuvaraj; Casilag, Fiordiligie; Kurth, Florian; Sander, Leif; Wendtner, Clemens-Martin; Hoechstetter, Manuela A.; Müller, Marcel A.; Sekul, Renate; Drosten, Christian; Stadler, Volker; Corman, Victor M.
Front. Immunol..
Mar 2021
10.3389/fimmu.2021.629185
The WHO declared the COVID-19 outbreak a public health emergency of international concern. The causative agent of this acute respiratory disease is a newly emerged coronavirus, named SARS-CoV 2, which originated in China in late 2019. Exposure to SARS‑CoV‑2 leads to multifaceted disease outcomes from asymptomatic infection to severe pneumonia, acute respiratory distress and potentially death. Understanding the host immune response is crucial for the development of interventional strategies. Humoral responses play an important role in defending viral infections and are therefore of particular interest. With the aim to resolve SARS-CoV-2-specific humoral immune responses at the epitope level, we screened clinically well-characterized sera from COVID-19 patients with mild and severe disease outcome using high-density peptide microarrays covering the entire proteome of SARS-CoV-2. Moreover, we determined the longevity of epitope-specific antibody responses in a longitudinal approach. Here we present IgG and IgA-specific epitope signatures from COVID-19 patients, which may serve as discriminating prognostic or predictive markers for disease outcome and/or could be relevant for intervention strategies

A new reliable and highly specific monoclonal antibody to detect the C‐terminal region of silencer of cytokine signaling 1

Weissinger, Stephanie E.; Zahn, Malena; Marienfeld, Ralf; Tessmer, Claudia; Moldenhauer, Gerhard; Möller, Peter
Eur J Haematol.
Mar 2021
10.1111/ejh.13620
Introduction SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading. Methods BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues. Results Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses. Conclusion This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein.

Signatures of antagonistic pleiotropy in a bacterial flagellin epitope

Parys, Katarzyna; Colaianni, Nicholas R.; Lee, Ho-Seok; Hohmann, Ulrich; Edelbacher, Natalie; Trgovcevic, Alen; Blahovska, Zuzana; Lee, Duhwa; Mechtler, Alexander; Muhari-Portik, Zsuzsanna; Madalinski, Mathias; Schandry, Niklas; Rodríguez-Arévalo, Isaac; Becker, Claude; Sonnleitner, Elisabeth; Korte, Arthur; Bläsi, Udo; Geldner, Niko; Hothorn, Michael; Jones, Corbin D.; Dangl, Jeffery L.; Belkhadir, Youssef
Cell Host & Microbe.
Mar 2021
10.1016/j.chom.2021.02.008
Immune systems respond to “non-self” molecules termed microbe-associated molecular patterns (MAMPs). Microbial genes encoding MAMPs have adaptive functions and are thus evolutionarily conserved. In the presence of a host, these genes are maladaptive and drive antagonistic pleiotropy (AP) because they promote microbe elimination by activating immune responses. The role AP plays in balancing the functionality of MAMP-coding genes against their immunogenicity is unknown. To address this, we focused on an epitope of flagellin that triggers antibacterial immunity in plants. Flagellin is conserved because it enables motility. Here, we decode the immunogenic and motility profiles of this flagellin epitope and determine the spectrum of amino acid mutations that drives AP. We discover two synthetic mutational tracks that undermine the detection activities of a plant flagellin receptor. These tracks generate epitopes with either antagonist or weaker agonist activities. Finally, we find signatures of these tracks layered atop each other in natural Pseudomonads.

Immunodominant B cell epitope in SARS-CoV-2 RBD comprises a B.1.351 and P.1 mutation hotspot: implications for viral spread and antibody escape

Santos, Keity Souza; Oliveira, Jamille Ramos; Machado, Rafael Rahal G.; Arcuri, Helen Andrade; Magawa, Jhosiene Yukari; Daher, Isabela Pazotti; Urbanski, Alysson Henrique; Schmitz, Gabriela Justamante Händel; Silva, Roberto Carlos Vieira; Durigon, Edison Luiz; Boscardin, Silvia Beatriz; Rosa, Daniela Santoro; Schechtman, Deborah; Nakaya, Helder I.; Cunha-Neto, Edecio; Gadermaier, Gabriele; Coelho, Verônica; Kalil, Jorge; Team, on behalf of COVID-19 SP-Brazil
Mar 2021
10.1101/2021.03.11.21253399

Recent SARS-CoV-2 variants pose important concerns due to their higher transmissibility (1) and escape (2) from previous infections or vaccine-induced neutralizing antibodies (nAb). The receptor binding domain (RBD) of the Spike protein is a major nAb target (3), but data on its B cell epitopes are still lacking. Using a peptide microarray, we identified an immunodominant epitope (S415-429) recognized by 68% of sera from 71 convalescent Brazilians infected with the ancestral variant. In contrast with previous studies, we have identified a linear IgG and IgA antibody binding epitope within the RBD. IgG and IgA antibody levels for this epitope positively correlated with nAb titers, suggesting a potential target of antibody neutralizing activity. Interestingly, this immunodominant RBD region harbors the mutation hotspot site K417 present in P.1 (K417T) and B.1.351 (K417N) variants. In silico simulation analyses indicate impaired RBD binding to nAb in both variants and that glycosylation in the B.1.351 417N could further hinder antibody binding as compared to the K417T mutation in P.1. This is in line with published data showing that nAb from either convalescents or anti-CoV-2 vaccinees are less effective towards B.1.351 than for P.1. Our data support the occurrence of immune pressure and selection involving this immunodominant epitope that may have critically contributed to the recent COVID-19 marked rise in Brazil and South Africa, and pinpoint a potential additional immune escape mechanism for SARS-CoV-2.

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