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

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Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome

Fathi, Anahita; Dahlke, Christine; Krähling, Verena; Kupke, Alexandra; Okba, Nisreen M. A.; Raadsen, Matthijs P.; Heidepriem, Jasmin; Müller, Marcel A.; Paris, Grigori; Lassen, Susan; Klüver, Michael; Volz, Asisa; Koch, Till; Ly, My L.; Friedrich, Monika; Fux, Robert; Tscherne, Alina; Kalodimou, Georgia; Schmiedel, Stefan; Corman, Victor M.; Hesterkamp, Thomas; Drosten, Christian; Loeffler, Felix F.; Haagmans, Bart L.; Sutter, Gerd; Becker, Stephan; Addo, Marylyn M.
Feb 2022
10.1101/2022.02.14.22270168
Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. A booster vaccination with MVA-MERS-S is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n=1) and the S2 subunit (n=3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials.

Scoping review of the applications of peptide microarrays on the fight against human infections

Vengesai, Arthur; Kasambala, Maritha; Mutandadzi, Hamlet; Mduluza-Jokonya, Tariro L.; Mduluza, Takafira; Naicker, Thajasvarie
PLoS ONE.
Jan 2022
10.1371/journal.pone.0248666
Introduction This scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis. Methods Electronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines. Results Ninety-five articles from 103 studies were included in the final data charting process. The majority (92. 0%) of the articles were published during 2010–2020 and were mostly from Europe (44.2%) and North America (34.7%). The findings were from the investigation of viral (45.6%), bacterial (32. 0%), parasitic (23.3%) and fungal (2. 0%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (77.7%) were related to mapping B-cell linear epitopes, 5.8% were on diagnostics, 5.8% reported on immunosignature characterisation and 8.7% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling. Conclusion The most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

Analysis of the Immune Response and Identification of Antibody Epitopes Against the Sigma C Protein of Avian Orthoreovirus Following Immunization with Live or Inactivated Vaccines

Dawe, W. H.; Kapczynski, D. R.; Linnemann, E. G.; Gauthiersloan, V. R.; Sellers, H. S.
Avian Diseases.
Jan 2022
10.1637/aviandiseases-D-22-99992

Epitope-Specific Anti-C1q Autoantibodies in Systemic Lupus Erythematosus

Kleer, Jessica S.; Rabatscher, Pascal A.; Weiss, Jessica; Leonardi, Joel; Vogt, Severin B.; Kieninger-Gräfitsch, Andrea; Chizzolini, Carlo; Huynh-Do, Uyen; Ribi, Camillo; Trendelenburg, Marten
Front. Immunol..
Jan 2022
10.3389/fimmu.2021.761395
Objective In patients with systemic lupus erythematosus (SLE) complement C1q is frequently targeted by autoantibodies (anti-C1q), that correlate best with active renal disease. Anti-C1q bind to largely unknown epitopes on the collagen-like region (CLR) of this highly functional molecule. Here we aimed at exploring the role of epitope-specific anti-C1q in SLE patients. Methods First, 22 sera of SLE patients, healthy controls and anti-C1q positive patients without SLE were screened for anti-C1q epitopes by a PEPperMAP® microarray, expressing CLR of C1q derived peptides with one amino acid (AA) shift in different lengths and conformations. Afterwards, samples of 378 SLE patients and 100 healthy blood donors were analyzed for antibodies against the identified epitopes by peptide-based ELISA. Relationships between peptide-specific autoantibodies and SLE disease manifestations were explored by logistic regression models. Results The epitope mapping showed increased IgG binding to three peptides of the C1q A- and three of the C1q B-chain. In subsequent peptide-based ELISAs, SLE sera showed significantly higher binding to two N-terminally located C1q A-chain peptides than controls (p < 0.0001), but not to the other peptides. While anti-C1q were associated with a broad spectrum of disease manifestations, some of the peptide-antibodies were associated with selected disease manifestations, and antibodies against the N-terminal C1q A-chain showed a stronger discrimination between SLE and controls than conventional anti-C1q. Conclusion In this large explorative study anti-C1q correlate with SLE overall disease activity. In contrast, peptide-antibodies are associated with specific aspects of the disease suggesting epitope-specific effects of anti-C1q in patients with SLE.

Protein microarrays for COVID-19 research: Biomarker discovery, humoral response, and vaccine targets

Acharjee, Arup; Barpanda, Abhilash; Ren, Jing; Yu, Xiaobo
Jan 2022
10.1201/9781003220787-6
Of all the technological interventions used to probe the COVID-19 biological sample, microarrays have provided unique information about the biology of SARS-CoV-2 infection in the greatest of detail. Protein microarrays are available in various formats such as protein microarray, antibody microarray, and peptide microarrays. These provide an attractive format to study host response against infection, with its straightforward sample preparation strategy and easy result analysis pipelines. Microarray technology either uses antibodies against hundreds of proteins to study host proteins or scans immunogenic peptides of the pathogen in a microarray panel of the pathogen proteome. It can be used to study the humoral immune response against antigenic proteins of the SARS-CoV-2 virus, host proteomic alterations due to the infection. The SARS-CoV-2 peptide array can be used for the accurate detection of antigenic determinants for vaccine design. This chapter summarizes the different types of protein and peptide microarray and their use in COVID-19 biomarker discovery, disease management, vaccine design, etc., for better management of COVID-19.

Induced Polyspecificity of Human Secretory Immunoglobulin A Antibodies: Is It Possible to Improve Their Ability to Bind Pathogens?

Gorshkova, Ekaterina N.; Pashova, Shina; Vasilenko, Ekaterina A.; Tchurina, Tatiana S.; Razzorenova, Elizaveta A.; Starkina, Olga V.; Dimitrova, Petya; Pashov, Anastas; Vassilev, Tchavdar Lubenov
Pharmacology.
Dec 2021
10.1159/000520343
Introduction: As has been shown previously, various protein-modifying agents can change the antigen-binding properties of immunoglobulins. However, induced polyspecificity of human secretory immunoglobulin A (sIgA) has not been previously characterized in detail. Methods: In the present study, human secretory immunoglobulin A (IgA) was exposed to buffers with acidic pH, to free heme, or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens was compared using Western blotting and enzyme-linked immunosorbent assay. The ability of these agents to modulate the antigen-binding properties of human sIgA toward a wide range of pathogen peptides was investigated using an epitope microarray. Results: We have shown that acidic pH, heme, and pro-oxidative ferrous ions influenced the binding of secretory IgA in opposite directions (either increasing or decreasing); however, the strongest effect was observed when using buffers with low pH. This fraction had the highest number of affected reactivities; most of them were increased and most of the new ones were toward common pathogens. Conclusions: Thus, it was shown that all investigated treatments can alter to some degree the antigen-binding of secretory IgA, but acidic pH has the most potentially beneficial effect by increasing binding to a largest number of common pathogens’ antigens.

Influenza‐associated thrombotic thrombocytopenic purpura: A report of two cases and a brief review of the literature

Onkarappa Mangala, Yashvin; Sweeney, Joseph D.
Vox Sanguinis.
Nov 2021
10.1111/vox.13227
Background and Objectives Thrombotic thrombocytopenic purpura (TTP) is often preceded by a recent history of an acute infection and influenza is the most implicated virus. Materials and Methods We identified two cases of TTP, which were preceded by influenza between 2010 and 2021. In one patient, we epitope mapped the binding specificity of antibodies using an overlapping peptide approach of the stalk protein of Influenza B and the cysteine-rich spacer domain (CRSD) of ADAMTS13. A literature search was performed for reports of influenza-associated TTP over the period 1980–2021. Results Two patients were identified in which TTP was preceded by influenza, one Influenza A and the other Influenza B. Epitope mapping of the latter’s plasma identified target epitopes in both the stalk protein of Influenza B and CRSD of ADAMTS13. The literature review revealed only seven case reports, all but one from Europe or Asia and associated with Influenza A. Severe ADAMTS13 deficiency was demonstrated in only four cases. Conclusion We report the first small case series of influenza-associated TTP. Moreover, it is the first case implicating Influenza B and a mechanism favouring polyclonal B-cell proliferation rather than molecular mimicry as the stimulus to form anti-ADAMTS13 auto-antibodies is suggested.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein

Wolfe, Monica; Webb, Sean; Chushak, Yaroslav; Krabacher, Rachel; Liu, Yi; Swami, Nathan; Harbaugh, Svetlana; Chávez, Jorge
Sci Rep.
Nov 2021
10.1038/s41598-021-01225-2
Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

Pras, Anita; Houben, Bert; Aprile, Francesco A.; Seinstra, Renée; Gallardo, Rodrigo; Janssen, Leen; Hogewerf, Wytse; Gallrein, Christian; De Vleeschouwer, Matthias; Mata-Cabana, Alejandro; Koopman, Mandy; Stroo, Esther; de Vries, Minke; Louise Edwards, Samantha; Kirstein, Janine; Vendruscolo, Michele; Falsone, Salvatore Fabio; Rousseau, Frederic; Schymkowitz, Joost; Nollen, Ellen A. A.
EMBO J.
Nov 2021
10.15252/embj.2020107568
While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Jaishankar, Dinesh; Cosgrove, Cormac; Ramesh, Prathyaya; Mahon, James; Shivde, Rohan; Dellacecca, Emilia R.; Yang, Shiayin F.; Mosenson, Jeffrey; Guevara-Patiño, José A.; Le Poole, I. Caroline
Cell Stress and Chaperones.
Sep 2021
10.1007/s12192-021-01229-x
Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70iQ435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70iQ435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70iQ435A in mice affects anti-tumor responses. We found that HSP70iQ435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70iQ435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70iQ435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70iQ435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.

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.

Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

Das, Aniruddha; Thapa, Pankaj; Santiago, Ulises; Shanmugam, Nilesh; Banasiak, Katarzyna; Dabrowska, Katarzyna; Nolte, Hendrik; Szulc, Natalia A.; Gathungu, Rose M.; Cysewski, Dominik; Krüger, Marcus; Dadlez, Michal; Nowotny, Marcin; Camacho, Carlos J.; Hoppe, Thorsten; Pokrzywa, Wojciech
Aug 2021
10.1101/2021.08.20.457118
The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

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