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

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Naturally acquired IgG responses to Plasmodium falciparum do not target the conserved termini of the malaria vaccine candidate Merozoite Surface Protein 2

Zerebinski, Julia; Margerie, Lucille; Han, Nan Sophia; Moll, Maximilian; Ritvos, Matias; Jahnmatz, Peter; Ahlborg, Niklas; Ngasala, Billy; Rooth, Ingegerd; Sjöberg, Ronald; Sundling, Christopher; Yman, Victor; Färnert, Anna; Plaza, David Fernando
Front. Immunol..
Dec 2024
10.3389/fimmu.2024.1501700
Introduction Malaria remains a significant burden, and a fully protective vaccine against Plasmodium falciparum is critical for reducing morbidity and mortality. Antibody responses against the blood-stage antigen Merozoite Surface Protein 2 (MSP2) are associated with protection from P. falciparum malaria, but its extensive polymorphism is a barrier to its development as a vaccine candidate. New tools, such as long-read sequencing and accurate protein structure modelling allow us to study the genetic diversity and immune responses towards antigens from clinical isolates with unprecedented detail. This study sought to better understand naturally acquired MSP2-specific antibody responses. Methods IgG responses against recombinantly expressed full-length, central polymorphic regions, and peptides derived from the conserved termini of MSP2 variants sequenced from patient isolates, were tested in plasma from travelers with recent, acute malaria and from individuals living in an endemic area of Tanzania. Results IgG responses towards full MSP2 and truncated MSP2 antigens were variant specific. IgG antibodies in the plasma of first-time infected or previously exposed travelers did not recognize the conserved termini of expressed MSP2 variants by ELISA, but they bound 13-amino acid long linear epitopes from the termini in a custom-made peptide array. Alphafold3 modelling suggests extensive structural heterogeneity in the conserved termini upon antigen oligomerization. IgG from individuals living in an endemic region, many who were asymptomatically infected, did not recognize the conserved termini by ELISA. Discussion Our results suggest that responses to the variable regions are critical for the development of naturally acquired immunity towards MSP2.

Anti-allodynic effect of intrathecal antibodies against macrophage-inducible C-type lectin in spinal nerve ligation model in rat

Kang, Dong Ho; Kim, Woong Mo; Bae, Hong Beom; Yang, Jihoon; Choi, Jeong Il
Heliyon.
Nov 2024
10.1016/j.heliyon.2024.e40694
*Introduction* Macrophage-inducible C-type lectin (Mincle) has emerged as a potential contributor to neuropathic pain induction and neuroinflammatory responses within the spinal cord. Moreover, evidence suggests a close association between toll-like receptor (TLR) and Mincle expression in myeloid cells. This study evaluated the effectiveness of Mincle antibodies in neuropathic pain and identified the epitope of these antibodies. In addition, the mode of interaction between Mincle and TLR inhibition was explored using isobolographic analysis. *Methods* Three different Mincle antibodies and a specific TLR4 inhibitor (TAK-242) were intrathecally administered, and mechanical allodynia was evaluated using the von Frey test in a rat model of spinal nerve ligation (SNL). Isobolographic analysis was conducted on the effect of combination of TAK-242 and Mincle Ab. Microarray analysis examined the specific region of Mincle targeted by the antibodies. *Results* All Mincle antibodies and TAK-242 significantly alleviated mechanical allodynia in a dose-dependent manner. However, the maximal possible effects (MPE) produced by the antibodies ranged widely from 37.1% to 91.8%, comparable to that of TAK-242 (88.7%). The combination of TAK-242 and the antibody with the highest MPE resulted in an additive interaction for their anti-allodynic effects. Epitope mapping revealed that each antibody targeted the extracellular domain, with epitope lengths ranging from 5 to 15 amino acids. *Conclusions* The current study demonstrates the anti-allodynic effect of Mincle antibodies and additive interaction with TLR4 inhibition in spinal nerve ligation model, suggesting the potential of blocking of Mincle signaling with its antibodies as a novel treatment strategy for neuropathic pain.

Vaccine-elicited and naturally elicited antibodies differ in their recognition of the HIV-1 fusion peptide

Reveiz, Mateo; Xu, Kai; Lee, Myungjin; Wang, Shuishu; Olia, Adam S.; Harris, Darcy R.; Liu, Kevin; Liu, Tracy; Schaub, Andrew J.; Stephens, Tyler; Wang, Yiran; Zhang, Baoshan; Huang, Rick; Tsybovsky, Yaroslav; Kwong, Peter D.; Rawi, Reda
Front. Immunol..
Nov 2024
10.3389/fimmu.2024.1484029
Broadly neutralizing antibodies have been proposed as templates for HIV-1 vaccine design, but it has been unclear how similar vaccine-elicited antibodies are to their naturally elicited templates. To provide insight, here we compare the recognition of naturally elicited and vaccine-elicited antibodies targeting the HIV-1 fusion peptide, which comprises envelope (Env) residues 512–526, with the most common sequence being AVGIGAVFLGFLGAA. Naturally elicited antibodies bound peptides with substitutions to negatively charged amino acids at residue positions 517–520 substantially better than the most common sequence, despite these substitutions rarely appearing in HIV-1; by contrast, vaccine-elicited antibodies were less tolerant of sequence variation, with no substitution of residues 512–516 showing increased binding. Molecular dynamics analysis and cryo-EM structural analysis of the naturally elicited ACS202 antibody in complex with the HIV-1 Env trimer with an alanine 517 to glutamine substitution suggested enhanced binding to result from electrostatic interactions with positively charged antibody residues. Overall, vaccine-elicited antibodies appeared to be more fully optimized to bind the most common fusion peptide sequence, perhaps reflecting the immunization with fusion peptide of the vaccine-elicited antibodies.

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.

Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

Chu, Xiaojie; Shin, Seungmin; Baek, Du-San; Zhang, Liyong; Conard, Alex; Shi, Megan; Kim, Ye-Jin; Adams, Cynthia; Hines, Maggie; Liu, Xianglei; Chen, Chuan; Sun, Zehua; Jelev, Dontcho V.; Mellors, John W.; Dimitrov, Dimiter S.; Li, Wei
mAbs.
Aug 2024
10.1080/19420862.2024.2387240
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63–69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice

Frische, Anders; Krogfelt, Karen Angeliki; Fomsgaard, Anders; Lassaunière, Ria
Vaccines.
Feb 2024
10.3390/vaccines12030218
An in-depth analysis of antibody epitopes following vaccination with different regimens provides important insight for developing future vaccine strategies. B-cell epitopes conserved across virus variants may be ideal targets for vaccine-induced antibodies and therapeutic drugs. However, challenges lie in identifying these key antigenic regions, and directing the immune system to target them. We previously evaluated the immunogenicity of two candidate DNA vaccines encoding the unmodified spike protein of either the SARS-CoV-2 Index strain or the Beta variant of concern (VOC). As a follow-on study, we characterized here the antibody binding profiles of three groups of mice immunized with either the DNA vaccine encoding the SARS-CoV-2 Index strain spike protein only, the Beta VOC spike protein only, or a combination of both as an antigen-heterologous prime-boost regimen. The latter induced an antibody response targeting overlapping regions that were observed for the individual vaccines but with additional high levels of antibody directed against epitopes in the SD2 region and the HR2 region. These heterologous-vaccinated animals displayed improved neutralization breadth. We believe that a broad-focused vaccine regimen increases neutralization breadth, and that the in-depth analysis of B-cell epitope targeting used in this study can be applied in future vaccine research.

ASFV epitope mapping by high density peptides microarrays

Desmet, Cloé; Coelho-Cruz, Bruna; Mehn, Dora; Colpo, Pascal; Ruiz-Moreno, Ana
Virus Research.
Jan 2024
10.1016/j.virusres.2023.199287
African swine fever (ASF) is an acute, highly contagious and deadly infectious disease. It is a threat to animal health with major potential economic and societal impact. Despite decades of ASF vaccine research, still some gaps in knowledge are hindering the development of a functional vaccine. Worth mentioning are gaps in understanding the mechanism of ASF infection and immunity, as well as the fact that – in case of this disease – virus proteins, so-called protective antigens, responsible for inducing protective immune responses in pigs are not identified yet. In this paper we elaborate on a methodology to identify protective antigens based on epitope mapping by microarray technology. High density peptide microarrays, combined with fluorescence scanning, have been used to analyze the interaction of peptide sequences of African swine fever virus (ASFV) proteins with antibodies present in inactivated serum from infected and healthy animals. The study evidenced ASFV proteins already under the radar for vaccine development, such as p54, and identified specific sequences in those proteins that may become the focus for future vaccine candidates. Such methodology is amenable to automation and high-throughput and may help developing better targeting for next generation vaccines.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein

Gardner, Thomas J.; Stein, Kathryn R.; Duty, J. Andrew; Schwarz, Toni M.; Noriega, Vanessa M.; Kraus, Thomas; Moran, Thomas M.; Tortorella, Domenico
Nat Commun.
Dec 2016
10.1038/ncomms13627
The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies’ epitope as an ‘antigenic hot spot’ critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Identification of novel antigens contributing to autoimmunity in cardiovascular diseases

Müller, Anna-Maria; Bockstahler, Mariella; Hristov, Georgi; Weiß, Christel; Fischer, Andrea; Korkmaz-Icöz, Sevil; Giannitsis, Evangelos; Poller, Wolfgang; Schultheiss, Heinz-Peter; Katus, Hugo A.; Kaya, Ziya
Clinical Immunology.
Dec 2016
10.1016/j.clim.2016.09.003
In myocarditis and dilated cardiomyopathy (DCM) patients the immune system may play an important role in disease progression. In this study, we aimed to identify new antigens as a target for autoimmune response that might play a crucial role in these diseases. Therefore, a peptide-array was used to investigate antibody binding profiles in patients with autoimmune myocarditis or DCM compared to healthy controls and thus to identify disease relevant antigens. To analyze the pathogenicity of the identified antigens, an experimental autoimmune myocarditis (EAM) model was used. Hereby, 3 peptide sequences, derived from myosin-binding-protein-C (MYBPC) fast-type, RNA-binding-protein 20 (RBM20), and dystrophin, showed pathogenic effects on the myocardium of mice. In summary, 3 potentially cardiopathogenic peptides (MYBPC fast-type, RBM20, dystrophin) were identified. Thus, this study could serve as a basis for future investigations aimed at determining further antigens leading to pathogenic effects on the myocardium of DCM as well as myocarditis patients.

Antibody Binding Studies Reveal Conformational Flexibility of the Bacillus cereus Non-Hemolytic Enterotoxin (Nhe) A-Component

Didier, A.; Dietrich, R.; Märtlbauer, E.
PLoS ONE.
Oct 2016
10.1371/journal.pone.0165135
The non-hemolytic enterotoxin complex (Nhe) is supposed to be the main virulence factor of B. cereus causing a diarrheal outcome of food poisoning. This tripartite toxin consists of the single components NheA, -B and -C all of them being necessary for maximum toxicity. In the past, research activities aiming to elucidate the mode-of-action of Nhe were mostly focused on the B- and C-component. In this study the generation of novel monoclonal antibodies (mAb) and their thorough characterization enabled the determination of key features for NheA. By the means of immunoaffinity chromatography it could be shown that NheA does not interact with -B and -C in solution. Additionally, the establishment of a highly sensitive sandwich-EIA now enables the detection of NheA in B. cereus supernatants down to 20 pg ml-1.Peptide-based epitope mapping in combination with partially deleted recombinant NheA fragments allowed the allocation of the binding regions for the three mAbs under study. Furthermore, by different EIA set-ups the conformational flexibility of NheA could be shown. For two of the antibodies under study different mechanisms of NheA neutralization were proven. Due to prevention of complete pore formation by one of the antibodies, NheA could be detected in an intermediate stage of the tripartite complex on the cell surface. Taken together, the results obtained in the present study allow a refinement of the mode-of-action for the Nhe toxin-complex.

A single amino acid substitution alter antigenicity of Glycosylated protein 4 of HP-PRRSV

Wang, Xinglong; Wang, Zhenbin; Xu, Hongyu; Biao, Xiang; Yang, Zengqi
Virol J.
Jul 2016
10.1186/s12985-016-0586-3
Background Porcine reproductive and respiratory syndrome (PRRS) is an important pig endemic disease in pork-producing countries worldwide. The etiology, porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by fast antigen variability. Glycosylated protein 4 (GP4) is a minor protein in PRRSV virion, but contributes to induce protective immune responses. However, the antigenic characterization of PRRSV GP4 and the role of the mutations in this protein in PRRSV evolution are not clear. Methods Peptides chip scanning and peptide based ELISA was used to analyze the antigenic characterization of HP-PRRSV GP4. A total of 142 peptides printed on a chip were used to reveal the antigen reaction characteristics of the HP-PRRSV. The reactions of these peptides with HP-PRRSV-specific pig serum were scanned and quantified using the software PepSlide® Analyzer by fluorescence intensity. The active reaction regions (AR) were identified based on the scanning results and then the amino acids (aa) sequences of AR(s) is aligned among PRRSV strains for further identify the key aa site(s) impact the antigenicity of the protein. Peptide based ELISA is then reacted with PRRSV positive sera derived from pig inoculated with different PRRSV strains for further analysis the role of specific amino acid in AR. Results The intensity plot was used to show the reactions of the peptides with PRRSV serum and it showed that enormously different response happened to various parts of GP4. The highest reaction intensity value reached 6401.5 against one peptide with the sequence DIKTNTTAASDFVVL. An AR from S29 to G56 was identified. Sequence alignment revealed various mutations in site 43 and possibly played an important role in this AR. Peptides ELISA reaction with sera from pigs inoculated with different PRRSV strain revealed that the change of aa in site 43 reduced the reaction of the peptide with PRRSV positive sera derived from pigs inoculated with the peptide related PRRSV strains. Conclusion In this study, one AR covering S29 to G56 was identified in GP4. The aa in site 43 play an important role in determining the antigenic character of GP4. The continual mutations (S → G → D → N) occurred in this site alter the antigenicity of PRRSV GP4.

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