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

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Identification of a Zika NS2B Epitope for Which Absence of IgG Response Is Associated with Severe Neurological Symptoms and the Design of a Biomarker Capable of Discriminatory Diagnostics Between Severe and Non4 Severe Clinical Phenotypes

Loeffler, Felix; Viana, Isabelle F. T.; Fischer, Nico; Coêlho, Danilo F.; Santos, Carolina; Purificacao Jr, Antonio; Araujo, Catarina; Leite, Bruno; Durães-Carvalho, Ricardo; Magalhaes, Thereza; Morais, Clarice; Tenório Cordeiro, Marli; Lins, Roberto; T. A. Marques, Ernesto; Jaenisch, Thomas
Mar 2021
10.26434/chemrxiv.14170439.v1
In this manuscript we describe the engineering of a biomarker for the diagnosis and prognosis of Zika-associated neurological disease. Although the causal association between congenital Zika virus (ZIKV) infection and neurological manifestations has been well documented in the recent years, biomarkers for proper diagnostic and disease outcome still remain to be defined. Combining high-density peptide array and multivariate analysis, we have identified an ZIKV epitope that is associated to a lack of IgG antibody response in patients with severe neurological symptoms. An engineered chimera was developed to discriminate between mild and severe clinical forms of the disease.

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
Mar 2021
10.1101/2021.03.04.433859
Abstract 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 Eighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010–2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% 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.

Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine

Farias, Leonardo P.; Vance, Gillian M.; Coulson, Patricia S.; Vitoriano-Souza, Juliana; Neto, Almiro Pires da Silva; Wangwiwatsin, Arporn; Neves, Leandro Xavier; Castro-Borges, William; McNicholas, Stuart; Wilson, Keith S.; Leite, Luciana C. C.; Wilson, R. Alan
amjor.
Mar 2021
10.3389/fimmu.2020.624613
The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 56 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyser software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritising vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Recent advances in the diagnosis of COVID-19: A bird’s eye view

Sharma, Bhawna; Shahanshah, Mohd FardeenHusain; Gupta, Sanjay; Gupta, Vandana
Expert Review of Molecular Diagnostics.
Mar 2021
10.1080/14737159.2021.1874354
Introduction: The COVID-19 pandemic is still escalating and has shaped an extraordinary and pressing need for rapid diagnostics with high sensitivity and specificity. Prompt diagnosis is the key to mitigate this situation. As several diagnostic tools for COVID-19 are already available and others are still under development, mandating a comprehensive review of the efficacy of existing tools and evaluate the potential of others.Areas Covered: Currently explored platforms for SARS-CoV-2 diagnostics and surveillance centered on qRT-PCR, RT-PCR, CRISPR, microarray, LAMP, lateral flow immunoassays, proteomics-based approaches, and radiological scans are overviewed and summarized in this review along with their advantages and downsides. A narrative literature review was carried out by accessing the freely available online databases to encapsulate the developments in medical diagnostics.Expert Opinion: An ideal detection method should be sensitive, specific, rapid, cost-effective, and should allow early diagnosis of the infection as near as possible to the point of care that could alter the current situation for the better. Medical diagnostics is a highly dynamic field as no diagnostic method available for SARS-CoV-2 detection offers a perfect solution and requires more attention and continuous R&D to challenge the present-day pandemic situation

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens

Harb, Jean; Mennesson, Nicolas; Lepetit, Cassandra; Fourny, Maeva; Louvois, Margaux; Bosseboeuf, Adrien; Allain-Maillet, Sophie; Decaux, Olivier; Moreau, Caroline; Tallet, Anne; Piver, Eric; Moreau, Philippe; Salle, Valéry; Bigot-Corbel, Edith; Hermouet, Sylvie
Feb 2021
10.3390/cells10020438
Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi

Latrofa, Maria Stefania; Palmisano, Giuseppe; Annoscia, Giada; Pierri, Ciro Leonardo; Chandrashekar, Ramaswamy; Otranto, Domenico
PLOS Neglected Tropical Diseases.
Feb 2021
10.1371/journal.pntd.0009027
Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

Zhang, Zhiqiang; Cottignie, Ines; Van Zeebroeck, Griet; Thevelein, Johan M.
Biochem J.
Jan 2021
10.1042/BCJ20200722
Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor–Sch9–TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor–Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region

Musicò, Angelo; Frigerio, Roberto; Mussida, Alessandro; Barzon, Luisa; Sinigaglia, Alessandro; Riccetti, Silvia; Gobbi, Federico; Piubelli, Chiara; Bergamaschi, Greta; Chiari, Marcella; Gori, Alessandro; Cretich, Marina
Vaccines.
Jan 2021
10.3390/vaccines9010035
A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

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.

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.

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.

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