Peptide Epitopes Research Articles

Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus (ASFV) is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects. To date, no licensed prophylactic vaccine exists. Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens. As such, there is a considerable requirement to understand the functional monoclonal antibodies (mAbs) and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines, therapeutics, and diagnostics. In this study, we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs, further screened a specific ASFV major capsid protein (p72) single-chain antibody and fused with an IgG Fc fragment (scFv-83-Fc), which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain. Using the scFv-83-Fc mAb, we selected a conserved epitope peptide (221MTGYKH226) of p72 retrieved from a phage-displayed random peptide library. Moreover, flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs, which indicated its potential application in vaccine and diagnostic reagent development. Overall, this study provided a valuable platform for identifying targets for ASFV vaccine development, as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Epitope mapping of Acinetobacter baumannii outer membrane protein W (OmpW) and laboratory study of an OmpW-derivative peptide

Outer membrane protein W (OmpW) is a less-known A. baumannii antigen with potential immunogenic properties. The epitopes of this protein are not well-identified yet. Therefore, in the present study, B- and T-cell epitopes of A. baumannii OmpW were found using comprehensive in silico and partially in vitro studies. The T-cell (both class-I and class-II) and B-cell (both linear and conformational) epitopes were predicted and screened through many bioinformatics approaches including the prediction of IFN-γ production, immunogenicity, toxicity, allergenicity, human similarity, and clustering. A single 15-mer epitopic peptide containing a linear B-cell and both classes of T-cell epitopes were found and used for further assays. For in vitro assays, patient- and healthy control-derived peripheral blood mononuclear cells were stimulated with the 15-mer peptide, Phytohemagglutinin, or medium alone, and cell proliferation and IFN-γ production assays were performed. The bioinformatics studies led to mapping OmpW epitopes and introducing a 15-mer peptide. In vitro assays to some extent showed its potency in cell proliferation but not in IFN-γ induction, although the responses were not very expressive and faced some questions/limitations. In general, in the current study, we mapped the most immunogenic epitopes of OmpW that may be used for future studies and also assayed one of these epitopes in vitro, which was shown to have an immunogenicity potential. However, the induced immune responses were not strong which suggests that the present peptide needs a series of biotechnological manipulations to be used as a potential vaccine candidate. More studies in this field are recommended.

Structural definition of HLA class II-presented SARS-CoV-2 epitopes reveals a mechanism to escape pre-existing CD4+ T cell immunity

CD4+ Tcells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ Tcell memory is finely poised at the level of peptide-HLA-II presentation.

Synthetic Peptide Antibodies as TNF‐α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF‐α in THP‐1 Derived Macrophages

AbstractTumor Necrosis Factor‐α (TNF‐α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF‐α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti‐TNF‐α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF‐α and preventing its binding to TNF‐α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP‐NGs). MIP‐NGs are synthetic antibodies obtained by nanomoulding the 3‐dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in‐house developed in silico rational approach, epitope peptides of TNF‐α were generated and ‘synthetic peptide antibodies’ were prepared. The resultant MIP‐NGs bind the template peptide and recombinant TNF‐α with high affinity and selectivity, and can block the binding of TNF‐α to its receptor. Consequently they were applied to neutralize pro‐inflammatory TNF‐α in the supernatant of human THP‐1 macrophages, leading to a downregulation of the secretion of pro‐inflammatory cytokines. Our results suggest that MIP‐NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost‐effective, are very promising as next generation TNF‐α inhibitors for the treatment of inflammatory diseases.

Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages.

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and 'synthetic peptide antibodies' were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide ("promiscuous" T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

T Cell Activating Thermostable Self-Assembly Nanoscaffold Tailored for Cellular Immunity Antigen Delivery.

Antigen delivery based on non-virus-like particle self-associating protein nanoscffolds, such as Aquifex aeolicus lumazine synthase (AaLS), is limited due to the immunotoxicity and/or premature clearance of antigen-scaffold complex resulted from triggering unregulated innate immune responses. Here, using rational immunoinformatics prediction and computational modeling, wescreen the T epitope peptides from thermophilic nanoproteins with the same spatial structure as hyperthermophilic icosahedral AaLS, and reassemble them into a novel thermostable self-assembling nanoscaffold RPT that can specifically activate T cell-mediated immunity. Tumor model antigen ovalbumin T epitopes and the severe acute respiratory syndrome coronavirus 2 receptor-binding domain are loaded onto the scaffold surface through the SpyCather/SpyTag system to construct nanovaccines. Compared to AaLS, RPT -constructed nanovaccines elicit more potent cytotoxic T cell and CD4+ T helper 1 (Th1)-biased immune responses, and generate less anti-scaffold antibody. Moreover, RPT significantly upregulate the expression of transcription factors and cytokines related to the differentiation of type-1 conventional dendritic cells, promoting the cross-presentation of antigens to CD8+ T cells and Th1 polarization of CD4+ T cells. RPT confers antigens with increased stability against heating, freeze-thawing, and lyophilization with almost no antigenicity loss. This novel nanoscaffold offers a simple, safe, and robust strategy for boosting T-cell immunity-dependent vaccine development.

Antibody responses to dietary antigens are accompanied by specific plasma cells in the infant thymus

Human infants develop IgG responses to dietary antigens during the first 2 years of life. Yet, the source of these antibodies is unclear. In previous studies we reported on the thymus as a unique functional niche for plasma cells (PCs) specific to environmental antigens. We sought to examine whether PCs specific to dietary antigens are detected in the infant thymus. We tested IgG reactivity to 112 food antigens and allergens in the serum of 20 neonates and infants using microarrays. The presence of PC-secreting IgG specific to the most prominent antigens was then assessed among thymocytes in the same cohort. Using an LC-MS proteomics approach, we looked for traces of these antigens in the thymus. Our studies first confirmed that cow's milk proteins are prevalent targets of serum IgG in early life. Subjects with the highest serum IgG titers to cow's milk proteins also harbored IgG-producing PCs specific to the same antigens in the thymic niche. Furthermore, we detected multiple peptide fragments of cow's milk antigens in the thymus. Lastly, we verified that both serum IgG and IgG secreted by thymic PCs recognized the peptide epitopes found in the thymus. Our studies reveal the presence of antibody-secreting PCs specific to common dietary antigens in the infant thymus. The presence of these antigens in the thymus suggested that activation and differentiation of specific PCs occurred in this organ. Further studies are now warranted to evaluate the possible implication of these cells in tolerance to dietary antigens.

Peptide immunotherapy for aeroallergens.

Background: Allergen specific immunotherapy (SIT) has been used for more than a century. Researchers have been working to improve efficacy and reduce the side effects. Objective: We have reviewed the literature about peptides immunotherapy for inhaled allergens. The mechanism of SIT is to induce regulatory T (Treg) cells and to reduce T helper (Th)2 cells to induce class switching from IgE to IgG and induce blocking antibodies to inhibit allergen binding of IgE. Methods: The relevant published literatures on the peptide SIT for aeroallergens have been searched on the medline. Results: Modification of allergens and routes of treatment has been performed. Among them, many researchers were interested in peptide immunotherapy. T-cell epitope peptide has no IgE epitope, that is able to bind IgE, but rather induces Treg and reduces Th2 cells, which was considered an ideal therapy. Results from cellular and animal model studies have been successful. However, in clinical studies, T-cell peptide immunotherapy has failed to show efficacy and caused side effects, because of the high effective rate of placebo and the development of IgE against T-cell epitope peptides. Currently, the modifications of IgE-allergen binding by blocking antibodies are considered for successful allergen immunotherapy. Conclusion: Newly developed hypoallergenic B cell epitope peptides and computational identification methods hold great potential to develop new peptide immunotherapies.

Chimeric antigen receptor T cell therapy for the treatment of systemic rheumatic diseases: a comprehensive review of recent literature.

Systemic rheumatoid diseases (SRDs) are autoimmune and inflammatory disorders that affect multiple organ systems, impacting patients' quality of life, and survival rates. Standard treatment requires continuous drug therapy and immunosuppression. Chimeric antigen receptor (CAR) T cell therapy has the potential to target and eliminate pathologically activated immune cells and re-establish tolerance in organs affected by dysregulated immunity, making them a promising treatment option for autoimmune diseases. In autoimmune diseases, CAR T cells have the advantage of being able to kill B cells effectively without the need for an accessory cell type. Additionally, CAR T cells targeting CD19 have shown promise in comprehensive B cell aplasia, preserving pre-existing humoral immunity, and specifically eliminating pathogenic B cells. CAR T cell therapy's limited use in SRDs is due to its inability to effectively target the various autoreactive lymphocytes present. Researchers are developing a universal CAR T cell therapy that detects and targets autoreactive lymphocytes using major epitope peptides, though further studies are required. Moreover, adoptive transfer of CAR-Tregs has shown promise for effectively reducing inflammation and treating autoimmunity. Through this exploration, the authors hope to provide a comprehensive understanding of the current state of research on this topic, identify areas for further study, and promote the advancement of CAR T cell therapy as a treatment option for SRDs.

Two peptides derivate from Acinetobacter baumannii outer membrane protein K as vaccine candidates: a comprehensive in silico study

BackgroundThe lack of appropriate vaccines is an obstacle to the effective management of A. baumannii infections. Peptide vaccines offer an attractive and promising preventive strategy against A. baumannii.ObjectiveIn this study, we identified specific T cell epitopes of A. baumannii outer membrane protein K (OMPK) using comprehensive bioinformatics and detailed molecular docking analysis.MethodsBoth class-I and class-II T cell epitopes of A. baumannii OMPK were predicted by three tools namely IEDB, SYFPEITHI, and ProPred. The predicted epitopes were shortlisted based on several analyses including prediction scoring, clustering, exclusion of human similarity, considering immunogenicity and cytokine production, and removal of toxic and/or allergen epitopes. The epitopic peptides with high prediction scores and appropriate properties containing both class-I and class-II T cell epitopes were selected. Two of these class I/II epitopic peptides were chosen for molecular docking studies and assessing their physicochemical properties as vaccine candidates.ResultsThe results showed many T-cell epitopes of OMPK that could be evaluated for possible immunogenicity. Two of these epitopes (containing both class-I and II epitopes) had high prediction scores, were predicted by several tools, attached to several HLAs, and had the best docking score. They had different physicochemical properties and were conserved among Acinetobacter species.DiscussionWe identified the A. baumannii OMPK high immunogenic class-I and class-II T cell epitopes and introduced two promising high immunogenic peptides as vaccine candidates. It is recommended to perform in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.

Improving Single-Molecule Antibody Detection Selectivity through Optimization of Peptide Epitope Presentation in OmpG Nanopore.

Outer membrane protein G (OmpG) is a monomeric porin found in Escherichia coli, which possesses seven flexible loops. OmpG has been engineered as a nanopore sensor, where its loops can host affinity epitopes for selective detection of biological molecules. In this study, we investigated various loop positions to incorporate a FLAG peptide antigen epitope in the most flexible loop 6 and tested the efficacy and sensitivity of these nanopore constructs in antibody detection. We observed an OmpG construct containing inserted FLAG sequence, which exhibited strong interaction with anti-FLAG antibodies in flow cytometry; however, it could not translate molecule interactions into a readable signal in current recordings. Further optimization of the peptide presentation strategy by replacing specific sections of loop 6 sequences with the FLAG tag created a construct capable of generating unique and distinct signals when interacting with various monoclonal or polyclonal anti-FLAG clones IgG antibodies in the mixture. The peptide display scheme demonstrated in this study can be generalized for the engineering of OmpG sensors, which can be used for screening and validating positive clones during antibody development, as well as for real-time quality control of cell cultures in monoclonal antibody production.

The position of Spy Tag/Catcher system in hepatitis B core protein particles affects the immunogenicity and stability of the synthetic vaccine

Presenting exogenous antigens on virus-like particles (VLPs) through “plug-and-display” decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on “plug-and-display” decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

295-OR: Mechanisms of Antigen-Specific Immunotherapy following Tolerance Induction to a CD4 Hybrid Insulin Peptide Epitope and the Impact on CD8 T Cell Dysfunction

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

Effect of polycation coating on the long-term pulsatile release of antigenic ESAT-61–20 peptide from PLGA nanoparticles

The development of novel vaccine formulations against tuberculosis is necessary to reduce the number of new cases worldwide. Polymeric nanoparticles offer great potential as antigen delivery and immunostimulant systems for such purposes. In the study, we have encapsulated the antigenic peptide epitope of ESAT-6 protein of M. tuberculosis into PLGA nanoparticles and coated these nanoparticles with the cationic polymer of quaternized poly(4-vinylpyridine) (QPVP) to obtain a positively charged system as a potential nasal vaccine prototype. The produced spherical nanoparticles had hydrodynamic diameters between 180 and 240 nm with a narrow size distribution. The non-coated nanoparticle exhibited a 3–phase in vitro release profile that was completed in more than 4 months. In this release study, 5% of the peptide was released in the first 6 h and the nanoparticle remained silent until the 70th day. Then, an additional 5% of the peptide was released in 45 days. After coating the nanoparticle with QPVP, the release periods and peptide amounts dramatically changed. The antigenic peptide-loaded nanoparticles coated with the polycation stimulated the macrophages in vitro to release more nitric oxide (NO) compared to the free peptide and non-coated nanoparticle, which reveals the immunostimulant activity of the produced nanoparticle systems. The produced non-coated nanoparticles with the prolonged pulsatile release of the antigenic peptide can be used in the development of single injection self-boosting vaccine formulations. By coating these nanoparticles, both the release profile and immunogenicity can be changed.

Antibody screening reveals antigenic proteins involved in Talaromyces marneffei and human interaction.

Talaromycosis is a fungal infection that generally affects immunocompromised hosts and is one of the most frequent systemic mycoses in HIV patients, especially in endemic areas such as Southeast Asia. Talaromyces marneffei, the causative agent of talaromycosis, grows as a mold in the environment but adapts to the human body and host niches by transitioning from conidia to yeast-like cells. Knowledge of the human host and T. marneffei interaction has a direct impact on the diagnosis, yet studies are still lacking. The morbidity and mortality rates are high in taloromycosis patients if the diagnosis and treatments are delayed. Immunogenic proteins are excellent candidates for developing detection tools. Previously, we identified antigenic proteins that were recognized by antibodies from talaromycosis sera. Three of these identified proteins have been previously characterized in detail, while the others have not been explored. To expedite the progress of antigen discovery, the complete list of antigenic proteins and their features was fully reported in this study. Functional annotation and Gene Ontology examination revealed that these proteins showed a high association with membrane trafficking. Further bioinformatics analyses were performed to search for antigenic protein characteristics, including functional domains, critical residues, subcellular localization, secretory signals, and epitope peptide sequences. Expression profiling of these antigenic encoding genes was investigated using quantitative real-time PCR. The results demonstrated that most genes were expressed at low levels in the mold form, but were highly upregulated in the pathogenic yeast phase, consistent with the antigenic role of these genes during the human-host interaction. Most transcripts accumulated in the conidia, suggesting a role during phase transition. The collection of all antigen-encoding DNA sequences described here is freely accessible at GenBank, which could be useful for the research community to develop into biomarkers, diagnostic tests, research detection tools, and even vaccines.

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This work developed a G‐quadruplexes‐based hydrogel consisting of guanine and peptide epitope to form a supramolecular hydrogel in the presence of metal cations. Using the metal ion‐responsive peptide epitope from the ion channel to replace sugar motif at N9 position of guanosine results in a novel nucleopeptide. The results show that the gelation time, the diameter of nanofibers, the anisotropic property, and the mechanical property of the nucleopeptide hydrogel can be simply controlled using metal cations. This work may provide a strategy for gluing pre‐existing fibers to form a hydrogel with tailored structures. More details are discussed in the article by Wang et al. on page 1727—1732.image

Detection of depression marker ASS1 in urine by gold nanoparticles based dual epitope-peptides imprinted sensor

Trace detection of argininosuccinate synthetase 1 (ASS1), a depression marker, in urine samples is difficult to achieve. In this work, a dual-epitope-peptides imprinted sensor for ASS1 detection in urine was constructed based on the high selectivity and sensitivity of the “epitope imprinting approach”. First, two cysteine-modified epitope-peptides were immobilized onto gold nanoparticles (AuNPs) deposited on a flexible electrode (ITO-PET) by gold-sulfur bonds (Au–S), then a controlled electropolymerization of dopamine was carried out to imprint the epitope peptides. After removing epitope-peptides, the dual-epitope-peptides imprinted sensor (MIP/AuNPs/ITO-PET) which with multiple binding sites for ASS1 was obtained. Compared with single epitope-peptide, dual-epitope-peptides imprinted sensor had higher sensitivity, which presented a linear range from 0.15 to 6000 pg ml−1 with a low limit of detection (LOD = 0.106 pg mL−1, S/N = 3). It had good reproducibility (RSD = 1.74%), repeatability (RSD = 3.60%), stability (RSD = 2.98%), and good selectivity, and the sensor had good recovery (92.4%–99.0%) in urine samples. This is the first highly sensitive and selective electrochemical assay for the depression marker ASS1 in urine, which is expected to provide help for the non-invasive and objective diagnosis of depression.

Generation of novel complete HLA class I monoallelic cell lines used in an MHC stabilization assay for neoantigen evaluation.

Immunogenic neoantigens derived from somatic mutations in cancer have been identified through clinical studies with the cloning of tumor-infiltrating T cells, and cancer driver gene mutation-derived epitopes have been reported; however, these are rare. At present, the validation of epitopes predicted in silico is difficult as human T-cell clonal diversity cannot be reproduced in vitro or in experimental animal models. To confirm the epitope peptides presented by human leukocyte antigen (HLA) class I molecules predicted in silico, biochemical methods such as major histocompatibility complex (MHC) stabilization assays and mass spectrometry-mediated identification have been developed based on HLA-A*02:01 monoallelic T2 cells and HLA-C*01:02 monoallelic LCL721.221 cells. Therefore, in the present study, to prevent confusion due to peptide cross-presentation among HLA molecules, HLA class I monoallelic B-cell clones were generated from the TISI cell line by knocking out HLA-ABC and TAP2, and knocking in HLA alleles. To explore cancer driver mutations as potential targets for immunotherapy, exome sequencing data from 5,143 patients with cancer enrolled in a comprehensive genome analysis project at the Shizuoka Cancer Center were used to identify somatic amino acid substituted mutations and the 50 most frequent mutations in five genes, TP53, EGFR, PIK3CA, KRAS and BRAF, were identified. Using NetMHC4.1, the present study predicted whether epitopes derived from these mutations are presented on major HLA-ABC alleles in Japanese individuals and synthesized 138 peptides for MHC stabilization assays. The authors also attempted to examine the candidate epitopes at physiological temperatures by using antibody clone G46-2.6, which can detect HLA-ABC, independent of β2-microglobulin association. In the assays, although the peptide-induced HLA expression levels were associated with the predicted affinities, the respective HLA alleles exhibited varying degrees of responsiveness, and unexpectedly, p53-mutant epitopes with predicted weak affinities exhibited strong responses. These results suggested that MHC stabilization assays using completely monoallelic HLA-expressing B-cell lines are useful for evaluating the presentation of neoantigen epitopes.