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

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Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

McNabb, Harrison J.; Cho, Eugene; Pitman, Mary; Rushton, Phillip S.; Mobley, David; Sjögren, Benita
Journal of Biological Chemistry.
Nov 2025
10.1016/j.jbc.2025.110757
Regulator of G protein signaling 2 (RGS2) negatively modulates signaling downstream of G protein–coupled receptors by accelerating GTP hydrolysis at Gα subunits of heterotrimeric G proteins. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease and asthma. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin–proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2–FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small-molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. We used a peptide array utilizing systematic residue substitution, combined with AlphaFold modeling and molecular dynamics simulations, to identify several amino acid changes that altered binding both positively and negatively. Finally, we experimentally confirmed our results in cells through coimmunoprecipitation and proteasomal inhibition, using full-length RGS2. Altogether, these results provide structural insights into RGS2–FBXO44 binding, which will aid in structure-guided drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein.

Severity-dependent IgG epitope profiling in COVID-19 reveals differential recognition of pathogen-derived antigens

Do Nascimento, Lais Alves; Machado, NicolleRakanidis; Borges, João Vitor Da Silva; Fagundes, Beatriz Oliveira; Bergamasco, Isabella Siuffi; Sgnotto, Fabio Da Ressureição; Bachi, André Luis Lacerda; Sato, Maria Notomi; Victor, Jefferson Russo
Front. Immunol..
Sep 2025
10.3389/fimmu.2025.1668223
Background The contribution of antibody-mediated responses to COVID – 19 outcomes remains unclear, particularly regarding cross-reactivity with unrelated pathogens. While co-infections are known to influence disease progression, the broader landscape of IgG reactivity during SARS-CoV-2 infection has not been systematically explored. Methods We employed a high-density peptide microarray containing 4,344 linear epitopes from 37 viruses, 27 bacteria, 17 parasites, and 8 fungi to characterize serum IgG repertoires from individuals with moderate (n = 39) or severe (n = 40) COVID – 19. Controls included pre-pandemic healthy donors and a pooled intravenous immunoglobulin (IVIg) formulation. Data analysis included intensity ranking, epitope mapping, and comparative analysis of mean signal intensities for each epitope between the COVID-Mod and COVID-Sev groups. Results COVID – 19 patients showed widespread IgG reactivity against diverse pathogens, with patterns differing by disease severity. Severe cases displayed broader and more intense reactivity, notably against hepatitis C virus (HCV), SARS-CoV-1, influenza A, Mycobacterium tuberculosis, and Plasmodium falciparum. Moderate cases showed preferential recognition of epitopes from HTLV-I, Neisseria meningitidis, and Trypanosoma cruzi. These findings suggest that SARS-CoV-2 infection modulates pre-existing humoral memory, possibly through epitope spreading or immune reprogramming. Conclusions SARS-CoV-2 infection reshapes the IgG epitope repertoire in a severity-dependent manner, extending to antigens from unrelated pathogens. This phenomenon may reflect underlying immune dysregulation or idiotype-driven interactions. Comprehensive profiling of pathogen-related IgG responses may reveal potential biomarkers of disease severity. This phenomenon may inform future investigations aimed at improving personalized management strategies for co-infected or immunocompromised patients.

Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

Boyd, Justin D.; Wang, Shixia; Lin, Hsiao-Wen; Hsieh, Yueh-Ting; Sun, Yu Shuang; Thibodeaux, Brett A.; Lu, Hanxin; Sahni, Jaya; Wiggins, Jonathan; Longo, Matthew S.; Brooks, Jeanne K.; Vroom, Madeline M.; Chang, Yi-Pin; Liu, Zhi; Ding, Shuang; Dodart, Jean-Cosme
Commun Med.
Apr 2025
10.1038/s43856-025-00870-2
Abstract **Background** The success of passive immunotherapies targeting Calcitonin gene-related peptide (CGRP) for managing migraine has prompted our efforts towards developing an active immunotherapy that induces the production of endogenous antibodies against CGRP. Achieving efficacious antibody titers via immunization could provide a more convenient and cost-effective treatment alternative to anti-CGRP monoclonal antibody (mAb) therapies. However, immunization against endogenous CGRP faces multiple challenges such as breaking immune tolerance, inducing sufficient antibody titers, and avoiding immune response-associated toxicity. **Methods** Synthetic peptide immunogens formulated in adjuvants were delivered intramuscularly. Serum samples were collected post immunization and used to measure antibody titers as well as for the isolation of antibodies specific to CGRP. Antibodies were characterized for their binding affinities and specificities. The capsaicin-induced increase in dermal blood flow model was used in rats for the assessment of the pharmacodynamic effect of immunization. **Results** Here we demonstrate that a peptide-based active immunotherapy designed to induce antibodies against CGRP promotes robust antibody titers across preclinical species. Characterization of the immune response strongly suggests that this peptide immunogen primarily stimulates a humoral response and only induced CGRP-specific antibodies. Antibodies produced by immunization are primarily IgG1 and demonstrate binding and activity potencies similar to marketed monoclonal antibodies against CGRP. Finally, immunization demonstrates in vivo efficacy in a rat pharmacodynamic model. **Conclusion** Our results strongly suggest that a peptide-based active immunotherapy against CGRP could provide an affordable and convenient therapeutic for the prevention of migraine.

A tumor-binding antibody with cross-reactivity to viral antigens

Campa, Michael J.; Gottlin, Elizabeth B.; Wiehe, Kevin; Patz, Edward F.
Cancer Immunol Immunother.
Feb 2025
10.1007/s00262-025-03975-8
**Background** We previously identified in non-small cell lung cancer (NSCLC) patients an autoantibody to complement factor H (CFH) that is associated with non-metastatic disease and longer time to progression in patients with stage I disease. A recombinant human antibody, GT103, was cloned from single B cells isolated from patients with the autoantibody. GT103 inhibits tumor growth and establishes an antitumor microenvironment. The anti-CFH autoantibody and GT103 recognize the epitope PIDNGDIT within the SCR19 domain of CFH. Here, we asked if this autoantibody could have originally arisen as a humoral response to a similar epitope in a viral protein from a prior infection. **Methods** Homologous viral peptides with high sequence identity to the core PIDNGDIT epitope sequence were identified and synthesized. NSCLC patient plasma containing anti-CFH autoantibodies were assayed by ELISA against these peptides. GT103 was assayed on a 4345-peptide pathogen microarray. **Results** Epitopes similar to the GT103 epitope are present in several viruses, including human metapneumovirus-1 (HMPV-1) that contains a sequence within attachment glycoprotein G that differs by one amino acid. Anti-CFH autoantibodies in NSCLC patient plasma weakly bound to an HMPV-1 peptide containing the epitope. GT103 cross-reacted with multiple viral epitopes on a peptide microarray, with the top hits being peptides in the human endogenous retrovirus-K polymerase (HERV-K pol) protein and measles hemagglutinin glycoprotein. GT103 bound the viral HMPV-1, HERV-K pol, and measles epitope peptides but with lower affinity compared to the GT103 epitope peptide. **Conclusion** These findings suggest that memory B cells against a viral target could have affinity matured to produce an antibody that recognizes a similar epitope on tumor cells and exhibits antitumor properties.

Investigation of Immunoreactivity Profiles and Epitope Landscape in Divergent COVID-19 Trajectories and SARS-CoV-2 Variants

Bihani, Surbhi; Ray, Arka; Borishetty, Dhanush; Tuckley, Chaitanya; Salkar, Akanksha; Acharjee, Arup; Shrivastav, Prithviraj; Shrivastav, Om; Shastri, Jayanthi; Agrawal, Sachee; Duttagupta, Siddhartha; Srivastava, Sanjeeva
J. Proteome Res..
Jan 2025
10.1021/acs.jproteome.4c00791
This study aimed to elucidate the complexity of the humoral immune response in COVID-19 patients with varying disease trajectories using a SARS-CoV-2 whole proteome peptide microarray chip. The microarray, containing 5347 peptides spanning the entire SARS-CoV-2 proteome and key variants of concern, was used to analyze IgG responses in 10 severe-to-recovered, 9 nonsevere-to-severe cases, and 10 control case (5 pre-pandemic and 5 SARS-CoV-2-negative) plasma samples. We identified 1151 IgG-reactive peptides corresponding to 647 epitopes, with 207 peptides being cross-reactive across 124 epitopes. Nonstructural protein 3 (nsp3) exhibited the highest number of total and unique epitopes, followed by the spike protein. nsp12 had the most number of cross-reactive epitopes. Peptides from the spike protein and nsps 2, 3, 5, and 13 were notably associated with recovery. Additionally, specific mutations in SARS-CoV-2 variants were found to alter peptide immunoreactivity, with some mutations (e.g., G142D, L452R, and N501Y) enhancing and others (e.g., R190S and E484 K) reducing immune recognition. These findings have critical implications for the development of diagnostics, vaccines, and therapeutics. Understanding the distribution of epitopes and the impact of viral mutations on antigenicity provides insights into immune evasion mechanisms, informing strategies for controlling COVID-19 and future coronavirus outbreaks.

High-throughput identification of immunoreactive peptides and corresponding proteins from Anaplasma platys and Ehrlichia canis using peptide microarray chips

Llanes, Alejandro; Madesh, Swetha; Brangulis, Kalvis; Rajeev, Sreekumari
Front Cell Infect Microbiol.
Jan 2025
10.3389/fcimb.2025.1671309
INTRODUCTION: Anaplasma platys and Ehrlichia canis are rickettsial pathogens infecting dogs, with a worldwide distribution. Both species are obligate intracellular pathogens and colonize bone marrow-derived cells, with coinfections frequently reported in dogs. Although E. canis immunodominant proteins have been thoroughly characterized, very few high-throughput studies have been conducted to identify immunogenic proteins from Anaplasma spp. In this study, we used a methodology based on peptide microarray chips to identify immunoreactive peptides, either shared or species-specific, in the complete theoretical proteomes of both pathogens. METHODS: B-cell epitopes were predicted in the corresponding proteins from both species and ranked for synthesis on the peptide microarrays. These microarrays were screened with serum samples from antibody-positive dogs, as well as negative control sera from unexposed dogs. Additionally, we assessed the feasibility of integrating evidence gathered at the level of individual peptides to identify potentially immunogenic proteins contributing to the patterns of immunoreactivity observed on microarrays. RESULTS: Screening of peptide microarrays resulted in complex antibody reactivity patterns against thousands of peptides. After discarding peptides with cross-reactivity to negative control sera, we identified over 1,200 immunoreactive peptides, including ~80 peptides shared between the two species with almost identical sequences. Despite screening linear peptides, we were able to identify proteins previously reported as immunodominant in E. canis, some of which contain predominantly conformational epitopes. DISCUSSION: Our results suggest that a high-throughput strategy based on peptide microarrays is an effective approach for the rapid identification of immunoreactive peptides and the underlying immunogenic proteins. This study provides a foundation for developing novel diagnostic tools and vaccine candidates against A. platys and E. canis, including potential combined or multivalent formulations targeting both pathogens.

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.

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