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

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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.

Bivalent chromatin accommodates survivin and BRG1/SWI complex to activate DNA damage response in CD4+ cells

Chandrasekaran, Venkataragavan; Andersson, Karin M. E.; Erlandsson, Malin; Li, Shuxiang; Olsson, Torbjörn Nur; Garcia-Bonete, Maria-Jose; Malmhäll-Bah, Eric; Johansson, Pegah; Katona, Gergely; Bokarewa, Maria I.
Cell Commun Signal.
Sep 2024
10.1186/s12964-024-01814-4
Background: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). Methods: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. Results: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. Conclusions: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.

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.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Schmidt, Ronny; Jacak, Jaroslaw; Schirwitz, Christopher; Stadler, Volker; Michel, Gerd; Marmé, Nicole; Schütz, Gerhard J.; Hoheisel, Jörg D.; Knemeyer, Jens-Peter
J. Proteome Res..
Jan 2011
10.1021/pr101070j

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