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

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Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen’s egg allergy in adults

Ehlers, Anna M.; Otten, Henny G.; Wierzba, Eva; Flügge, Ulrike; Le, Thuy-My; Knulst, André C.; Suer, Waltraud
Sep 2020
10.1111/cea.13730
Background Although hen’s egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffers from a persistent or newly-onset hen’s egg allergy, making accurate diagnosis in adults necessary. However, sensitisation to hen’s egg extracts, components and linear epitopes are solely studied in children. Methods Hen’s egg allergic (n=16) and tolerant (n=20) adults were selected by sensitisation towards recombinant components rGal d 1 and/or 3. Sensitisation profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterisation of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. Results Overall, sIgE titres against hen’s egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitisation to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. Conclusion and Clinical Relevance Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen’s egg allergic adults with objective symptoms.

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F
J. Proteome Res..
Sep 2020
10.1021/acs.jproteome.0c00484
Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 500 000 deaths globally. Outbreaks caused by viruses such as SARS-CoV-2, HIV, Ebola, influenza, and Zika have increased over the last decade, underlining the urgent need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides. Using these highly diverse libraries, covering tens of thousands of peptides, allow for the in-depth analysis of antibody signatures in a multiplexed, high-throughput fashion. In this review, we highlight synthesis platforms that facilitate fast and highly flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines, to quickly encounter pandemic threats.

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

Fu, Yingying; Cao, Rui; Schäfer, Miriam; Stephan, Sonja; Braspenning-Wesch, Ilona; Schmitt, Laura; Bischoff, Ralf; Müller, Martin; Schäfer, Kai; Vinzón, Sabrina E; Rösl, Frank; Hasche, Daniel
Aug 2020
10.7554/eLife.57626
Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.

Molecular and Serological Tests for COVID-19. A Comparative Review of SARS-CoV-2 Coronavirus Laboratory and Point-of-Care Diagnostics

Kubina, Robert; Dziedzic, Arkadiusz
Jun 2020
10.3390/diagnostics10060434
Validated and accurate laboratory testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a crucial part of the timely management of Coronavirus Disease 2019 (COVID-19) disease, supporting the clinical decision-making process for infection control at the healthcare level and detecting asymptomatic cases. This would facilitate an appropriate treatment, a prompt isolation and consequently deceleration of the pandemic. Various laboratory tests can identify the genetic material of SARS-CoV-2 that causes COVID-19 in specimens, or specific anti-viral antibodies in blood/serum. Due to the current pandemic situation, a development of point-of-care diagnostics (POCD) allows us to substantially accelerate taking clinical decisions and implement strategic planning at the national level of preventative measures. This review summarizes and compares the available POCD and those currently under development, including quantitative reverse transcription PCR (RT-qPCR), serology immunoassays (SIAs) and protein microarray method (PMM) designed for standard and rapid COVID-19 diagnosis.

Epitopes of Naturally Acquired and Vaccine‐induced Anti‐Ebola Virus Glycoprotein Antibodies in Single Amino Acid Resolution

Heidepriem, Jasmin; Krähling, Verena; Dahlke, Christine; Wolf, Timo; Klein, Florian; Addo, Marylyn M.; Becker, Stephan; Loeffler, Felix F.
Biotechnol. J..
May 2020
10.1002/biot.202000069
The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti-EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, the analysis of serum samples from an EBOV vaccine trial with the recombinant vesicular stomatitis virus-Zaire ebolavirus (rVSV-ZEBOV) and an Ebola virus disease survivor, using high-density peptide arrays, is presented. In this proof-of-principle study, distinct IgG and IgM antibodies binding to different epitopes of EBOV GP is detected: By mapping the whole GP as overlapping peptide fragments, new epitopes and confirmed epitopes from the literature are found. Furthermore, the highly selective binding epitope of a neutralizing monoclonal anti-EBOV GP antibody could be validated. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development.

Autoantibody Signature in Cardiac Arrest

Maguy, Ange; Tardif, Jean-Claude; Busseuil, David; Ribi, Camillo; Li, Jin
Circulation.
Apr 2020
10.1161/CIRCULATIONAHA.119.044408
Background: Cardiac arrest is a tragic event that causes one death roughly every 90 seconds worldwide. Survivors generally undergo a work-up to identify the etiology of arrest. However, 5-10% of cardiac arrest remain unexplained. As cardiac arrhythmias mostly underlie cardiac arrest and increasing evidence strongly supports the involvement of autoantibodies in arrhythmogenesis, a large-panel autoantibody screening was performed in cardiac arrest patients. Methods: This is an observational, cross-sectional study of patients from the Montreal Heart Institute (MHI) hospital cohort, a single center registry of participants. A peptide microarray was designed to screen for IgG targeting epitopes from all known cardiac ion channels with extracellular domains. Plasma samples from 23 patients with unexplained cardiac arrest were compared to 22 cardiac arrest cases of ischemic origin and a group of 29 age-, sex- and BMI-matched healthy subjects. The false discovery rate (FDR), LASSO logistic regression and random forest methods were jointly carried out to find significant differential IgG responses. Results: The autoantibody against the pore domain of the L-type voltage-gated calcium channel (Ca v 1.2) was consistently identified as a biomarker of idiopathic cardiac arrest (P=0.002, FDR=0.007, classification accuracies ≥0.83). Functional studies on human induced pluripotent stem cell-derived cardiomyocytes demonstrated that the anti-Ca v 1.2 IgG purified from patients with idiopathic cardiac arrest is proarrhythmogenic by reducing the action potential duration through calcium channel inhibition. Conclusions: The present report addresses the concept of autoimmunity and cardiac arrest. Hitherto unknown autoantibodies targeting extracellular sequences of cardiac ion channels were detected. Moreover, the study identified an autoantibody signature specific to patients with cardiac arrest.

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.

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).

Discovery of putative breast cancer antigens using an integrative platform of genomics-driven immunoproteomics

Qendro, Veneta; Lundgren, Deborah H.; Palczewski, Samuel; Hegde, Poornima; Stevenson, Christina; Perpetua, Laurie; Latifi, Ardian; Merriman, Jesse; Bugos, Grace; Han, David K.
Proteomics.
Aug 2018
10.1002/pmic.201600318
Recent advances in cancer immuno-therapeutics such as checkpoint inhibitors, chimeric antigen-receptor T cells, and tumor infiltrating T cells (TIL) are now significantly impacting cancer patients in a positive manner. Although very promising, reports indicate no more than 25% of cases result in complete remission. One of the limitations of these treatments is the identity of putative cancer antigens in each patient, as it is technically challenging to identify cancer antigens in a rapid fashion. Thus, identification of cancer antigens followed by targeted treatment will increase the efficacy of cancer immunotherapies. To achieve this goal, a combined technologies platform of deep genomic sequencing and personalized immune assessment was devised, termed Genomics Driven Immunoproteomics (GDI). Using this technological platform, we report the discovery of 149 tumor antigens from human breast cancer patients. Significant number of these putative cancer antigens arise from single nucleotide variants (SNVs), as well as insertions and deletions that results into frame-shift mutations. We propose a general model of anti-cancer immunity and suggest that the GDI platform may help identify patient-specific tumor antigens in a timely fashion for precision immunotherapies.

Circulating GRP78 antibodies from ovarian cancer patients: a promising tool for cancer cell targeting drug delivery system?

Van Hoesen, Kylie; Meynier, Sonia; Ribaux, Pascale; Petignat, Patrick; Delie, Florence; Cohen, Marie
Oncotarget.
Dec 2017
10.18632/oncotarget.22412
Glucose-regulated protein 78 (GRP78) is a chaperone protein that has a high frequency in tumor cells. Normally it is found in the endoplasmic reticulum to assist in protein folding, but under cellular stress, GRP78 influences proliferative signaling pathways at the cell surface. The increased expression elicits autoantibody production, providing a biomarker of ovarian cancer, as well as other types of cancer. This study aims to determine the epitope recognition of GRP78 autoantibodies isolated from serum of ovarian cancer patients and use the identified antibodies to design new drug delivery systems to specifically target cancer cells. We first confirmed that the membrane GRP78 levels are increased in ovarian cancer cells and positively correlate with proliferation. However, the level of circulating GRP78 autoantibodies did not correlate with membrane GRP78 expression in ovarian cancer cells and was lower, although not significantly, compared to control patients. We then determined the epitope recognition of GRP78 autoantibodies and showed that treatment with paclitaxel-loaded nanoparticles coated with anti-GRP78 antibodies significantly decreased tumor development in chick embryo culture of ovarian cancer cell tumors compared to paclitaxel treatment alone. This evidence suggests that nanoparticle drug delivery systems coupled with antibodies against GRP78 has potential as a powerful therapy against ovarian cancer.

Development and analytical performance of a new ARCHITECT automated dipeptidyl peptidase-4 immunoassay

Hemken, Philip M.; Jeanblanc, Nicolette M.; Rae, Tracey; Brophy, Susan E.; Datwyler, Maria J.; Xu, Ying; Manetz, T. Scott; Vainshtein, Inna; Liang, Meina; Xiao, Xiaodong; Chowdhury, Partha S.; Chang, Chien-ying; Streicher, Katie; Greenlees, Lydia; Ranade, Koustubh; Davis, Gerard J.
Practical Laboratory Medicine.
Dec 2017
10.1016/j.plabm.2017.10.003
Background Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations. Methods We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma. Results The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation) was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics. Conclusion These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration.

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Desombere, Isabelle; Mesalam, Ahmed Atef; Urbanowicz, Richard A.; Van Houtte, Freya; Verhoye, Lieven; Keck, Zhen-Yong; Farhoudi, Ali; Vercauteren, Koen; Weening, Karin E.; Baumert, Thomas F.; Patel, Arvind H.; Foung, Steven K.H.; Ball, Jonathan; Leroux-Roels, Geert; Meuleman, Philip
Antiviral Research.
Dec 2017
10.1016/j.antiviral.2017.10.015
Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

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