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

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High-resolution mapping of linear epitopes from LiNTPDase2: Advancing leishmaniasis detection using optimized protein and peptide antigens

Castro, Raissa Barbosa De; Badaró De Moraes, João Victor; De Souza, Anna Cláudia Alves; Favarato, Evandro Silva; Voorwald, Fabiana Azevedo; Dos Santos, Fabiane Matos; Bressan, Gustavo Costa; Vasconcellos, Raphael De Souza; Fietto, Juliana Lopes Rangel
Diagnostic Microbiology and Infectious Disease.
Oct 2024
10.1016/j.diagmicrobio.2024.116448
Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.

Identification of Schistosoma haematobium and Schistosoma mansoni linear B-cell epitopes with diagnostic potential using in silico immunoinformatic tools and peptide microarray technology

Vengesai, Arthur; Manuwa, Marble; Midzi, Herald; Mandeya, Masimba; Muleya, Victor; Mujeni, Keith; Chipako, Isaac; Mduluza, Takafira
PLoS Negl Trop Dis.
Aug 2024
10.1371/journal.pntd.0011887
Introduction: Immunoinformatic tools can be used to predict schistosome-specific B-cell epitopes with little sequence identity to human proteins and antigens other than the target. This study reports an approach for identifying schistosome peptides mimicking linear B-cell epitopes using in-silico tools and peptide microarray immunoassay validation. Method: Firstly, a comprehensive literature search was conducted to obtain published schistosome-specific peptides and recombinant proteins with the best overall diagnostic performances. For novel peptides, linear B-cell epitopes were predicted from target recombinant proteins using ABCpred, Bcepred and BepiPred 2.0 in-silico tools. Together with the published peptides, predicted peptides with the highest probability of being B-cell epitopes and the lowest sequence identity with proteins from human and other pathogens were selected. Antibodies against the peptides were measured in sera, using peptide microarray immunoassays. Area under the ROC curve was calculated to assess the overall diagnostic performances of the peptides. Results: Peptide AA81008-19-30 had excellent and acceptable diagnostic performances for discriminating S. mansoni and S. haematobium positives from healthy controls, with AUC values of 0.8043 and 0.7326 respectively for IgG. Peptides MS3_10186-123-131, MS3_10385-339-354, SmSPI-177-193, SmSPI-379-388, MS3-10186-40-49 and SmS-197-214 had acceptable diagnostic performances for discriminating S. mansoni positives from healthy controls with AUC values ranging from 0.7098 to 0.7763 for IgG. Peptides SmSPI-359-372, Smp126160-438-452 and MS3 10186-25-41 had acceptable diagnostic performances for discriminating S. mansoni positives from S. mansoni negatives with AUC values of 0.7124, 0.7156 and 0.7115 respectively for IgG. Peptide MS3-10186-40-49 had an acceptable diagnostic performance for discriminating S. mansoni positives from healthy controls, with an AUC value of 0.7413 for IgM. Conclusion: One peptide with a good diagnostic performance and nine peptides with acceptable diagnostic performances were identified using the immunoinformatic approach and peptide microarray validation. There is need for evaluation of the peptides with true negatives and a good standard positive reference.

Alzheimer’s disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels

Sim, Kyu-Young; An, Jaekyeung; Bae, So-Eun; Yang, Taewoo; Ko, Gwang-Hoon; Hwang, Jeong-Ryul; Choi, Kyu Yeong; Park, Jung Eun; Lee, Jung Sup; Kim, Byeong C.; Lee, Kun Ho; Park, Sung-Gyoo
Journal of Infection and Public Health.
Jul 2024
10.1016/j.jiph.2024.05.050
*Background* Alzheimer’s disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. *Methods* To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95–8 cell line. *Results* In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95–8 cells. *Conclusion* Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value

Grąźlewska, Weronika; Holec-Gąsior, Lucyna; Sołowińska, Karolina; Chmielewski, Tomasz; Fiecek, Beata; Contreras, Marinela
ACS Infect. Dis..
Oct 2023
10.1021/acsinfecdis.3c00258

Linear epitope mapping in the E and NS1 proteins of dengue and Zika viruses: Prospection of peptides for vaccines and diagnostics

Aquino, Victor Hugo; Fumagalli, Marcilio J.; Silva, Angélica; De Moura Negrini, Bento Vidal; Rojas, Alejandra; Guillen, Yvalena; Bernal, Cynthia; Figueiredo, Luiz Tadeu Moraes
PLoS ONE.
Oct 2023
10.1371/journal.pone.0292451
The arrival of the Zika virus (ZIKV) in dengue virus (DENV)-endemic areas has posed challenges for both differential diagnosis and vaccine development. Peptides have shown promise in addressing these issues. The aim of this study was to identify the linear epitope profile recognized by serum samples from dengue and Zika patients in the E and NS1 proteins of DENV and ZIKV. This cross-sectional study included individuals of all ages with laboratory-confirmed DENV and ZIKV infections, who were selected through convenience sampling. The serum samples from dengue and Zika patients detected epitopes evenly distributed across the viral proteins in a peptide microarray platform. However, several epitopes were located within “epitope hotspots”, characterized by clusters of peptides recognized in more than 30% of the sub-arrays analyzed using individual or pooled serum samples. The serum samples from dengue and Zika patients showed a high level of cross-reactivity with peptides in the DENV and ZIKV proteins. Analysis using an additional peptide microarray platform, which contained peptides selected based on the results of the initial screening, revealed that two DENV and one ZIKV peptide, highly specific to their related viruses, were located within the epitope hotspots; however, they presented low detection rates (32.5, 35.0, and 28.6%, respectively). In addition, two DENV peptides detected at similarly high rates by both dengue and Zika patients were also found within the epitope hotspots. These hotspots contain several immunodominant epitopes that are recognized by a larger number of individuals when compared to 15-amino acid (aa) sequence peptides. Thus, epitope hotspots may have greater potential to serve as antigens in diagnostic tests and vaccine development than peptides composed of only 15 amino acids.

Antigen discovery by bioinformatics analysis and peptide microarray for the diagnosis of cystic echinococcosis

Batisti Biffignandi, Gherard; Vola, Ambra; Sassera, Davide; Najafi-Fard, Saeid; Gomez Morales, Maria Angeles; Brunetti, Enrico; Teggi, Antonella; Goletti, Delia; Petrone, Linda; Tamarozzi, Francesca
PLoS Negl Trop Dis.
Apr 2023
10.1371/journal.pntd.0011210
Background Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected zoonosis. Its diagnosis relies on imaging, supported by serology, while only imaging is useful for staging and follow-up. Since diagnostic tools and expertise are not widely available, new accurate and easily implementable assays for the diagnosis and follow-up of CE are highly needed. Methodology/Principal Findings We aimed to identify new E . granulosus antigens through a bioinformatics selection applied to the parasite genome, followed by peptide microarray screening and validation in ELISA, using independent panels of sera from patients with hepatic CE and clinically relevant controls. From 950 proteins selected in silico , 2,379 peptides were evaluated by microarray for IgG reactivity and eight candidates selected for validation. Reactivity to one peptide was significantly higher in the CE group (p = 0.044), but had suboptimal diagnostic accuracy. Conclusions/Significance Here we performed bioinformatics analysis and peptide microarray for antigen discovery, useful for the diagnosis of CE. Eight candidates were selected and validated. Reactivity to one peptide associated to CE but had suboptimal diagnostic accuracy. Importantly, the database developed in this study may be used to identify other antigenic candidates for CE diagnosis and follow-up.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis

Nessler, Jasmin Nicole; Tipold, Andrea
PLoS ONE.
Apr 2023
10.1371/journal.pone.0284010
Steroid responsive meningitis arteritis (SRMA) is an aberrant Th2-mediated systemic inflammatory disease in dogs. The etiopathogenesis still remains unclear as no triggering pathogen or autoantigen could be found so far. Hypothesis . Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. Methods . The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Results . Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. Discussion . IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments

Severino, Rita; Moreno-Paz, Mercedes; Puente-Sánchez, Fernando; Sánchez-García, Laura; Risso, Valeria A.; Sanchez-Ruiz, Jose M.; Cabrol, Nathalie; Parro, Victor
Anal. Chem..
Mar 2023
10.1021/acs.analchem.2c05386

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests

Prudencio, Carlos Roberto; Gomes da Costa, Vivaldo; Rocha, Leticia Barboza; Costa, Hernan Hermes Monteiro; Orts, Diego José Belato; da Silva Santos, Felipe Rocha; Rahal, Paula; Lino, Nikolas Alexander Borsato; da Conceição, Pâmela Jóyce Previdelli; Bittar, Cintia; Machado, Rafael Rahal Guaragna; Durigon, Edison Luiz; Araujo, João Pessoa; Polatto, Juliana Moutinho; da Silva, Miriam Aparecida; de Oliveira, Joyce Araújo; Mitsunari, Thais; Pereira, Lennon Ramos; Andreata-Santos, Robert; de Souza Ferreira, Luís Carlos; Luz, Daniela; Piazza, Roxane Maria Fontes
Viruses.
Feb 2023
10.3390/v15030654
Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

IgE and IgG4 Epitopes of Dermatophagoides and Blomia Allergens before and after Sublingual Immunotherapy

Figo, Daniele Danella; Cordeiro Macedo, Priscilla Rios; Gadermaier, Gabriele; Remuzgo, Cesar; Castro, Fábio Fernandes Morato; Kalil, Jorge; Galvão, Clovis Eduardo Santos; Santos, Keity Souza
IJMS.
Feb 2023
10.3390/ijms24044173
Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

Exploring the Immunodominant Epitopes of SARS-CoV-2 Nucleocapsid Protein as Exposure Biomarker

Vashisht, Kapil; Goyal, Bharti; Pasupureddy, Rahul; Na, Byoung-Kuk; Shin, Ho-Joon; Sahu, Dibakar; De, Sajal; Chakraborti, Soumyananda; Pandey, Kailash C
Feb 2023
10.7759/cureus.34827

Reactivity Graph Yields Interpretable IgM Repertoire Signatures as Potential Tumor Biomarkers

Ferdinandov, Dilyan; Kostov, Viktor; Hadzhieva, Maya; Shivarov, Velizar; Petrov, Peter; Bussarsky, Assen; Pashov, Anastas Dimitrov
IJMS.
Jan 2023
10.3390/ijms24032597
Combining adaptive and innate immunity induction modes, the repertoire of immunoglobulin M (IgM) can reflect changes in the internal environment including malignancies. Previously, it was shown that a mimotope library reflecting the public IgM repertoire of healthy donors (IgM IgOme) can be mined for efficient probes of tumor biomarker antibody reactivities. To better explore the interpretability of this approach for IgM, solid tumor-related profiles of IgM reactivities to linear epitopes of actual tumor antigens and viral epitopes were studied. The probes were designed as oriented planar microarrays of 4526 peptide sequences (as overlapping 15-mers) derived from 24 tumor-associated antigens and 209 cancer-related B cell epitopes from 30 viral antigens. The IgM reactivity in sera from 21 patients with glioblastoma multiforme, brain metastases of other tumors, and non-tumor-bearing neurosurgery patients was thus probed in a proof-of-principle study. A graph representation of the binding data was developed, which mapped the cross-reactivity of the mixture of IgM (poly)specificities, delineating different antibody footprints in the features of the graph—neighborhoods and cliques. The reactivity graph mapped the major features of the IgM repertoire such as the magnitude of the reactivity (titer) and major cross-reactivities, which correlated with blood group reactivity, non-self recognition, and even idiotypic specificities. A correlation between an aspect of this image of the IgM IgOme, namely, small cliques reflecting rare self-reactivities and the capacity of subsets of the epitopes to separate the diagnostic groups studied was found. In this way, the graph representation helped the feature selection in its filtering step and provided reduced feature sets, which, after recursive feature elimination, produced a classifier containing 51 peptide reactivities separating the three diagnostic groups with an unexpected efficiency. Thus, IgM IgOme approaches to repertoire studies is greatly augmented when self/viral antigens are used and the data are represented as a reactivity graph. This approach is most general, and if it is applicable to tumors in immunologically privileged sites, it can be applied to any solid tumors, for instance, breast or lung cancer.

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