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In this study, hydrolysates of Rana spinosa meat were purified and characterized, and combined with molecular docking to screen potential immunomodulatory peptides and explore their activities and mechanisms of action. The results showed that 582 peptides were identified from the hydrolysates, and three novel immunomodulatory peptides, GIHETTYNS (1020.4512 Da), IADRMQKE (989.4964 Da), and IVRDIKEK (999.6077 Da), were obtained by molecular docking. These peptides significantly increased the proliferative activity of RAW264.7 cells and accelerated its cell cycle proceeding, promoted the production of NO, IL-6, and TNF-α, and enhanced ROS levels. The molecular docking analysis revealed that immunomodulatory peptides bound to the key regions of TLR4/MD-2 by hydrogen bonds and hydrophobic interactions, and the common sites of action were LYS A:458, ARG A:434, and ARG D: 90. Furthermore, these immunomodulatory peptides had favorable safety and stability properties in silico analysis. These novel peptides are expected to be new natural materials for immunomodulators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ochratoxin A (OTA) is a common food contaminant and poses a significant threat to human health, which requires rigorous monitoring. Mimotope peptides (MPs) are commonly used as non-toxic alternatives to toxic small molecules in eco-friendly immunoassays. Herein, with an anti-OTA nanobody as the target protein, cyclic 7-mer MPs of OTA were screened using phage display and immunomagnetic separation. The phage MPs (PMP) with the highest sensitivity and its alkaline phosphatase-tagged MP fusion (ALP-MP) were used to develop a PMP-based chemiluminescent immunoassay (PMP-CLIA) and an ALP-MP-based CLIA (AMP-CLIA). After optimization, PMP-CLIA and AMP-CLIA exhibited a limit of detection of 0.128 ng/mL and 0.232 ng/mL. Good accuracy and selectivity were confirmed for both CLIAs by recovery experiments and cross-reactions. Moreover, they were validated by high performance liquid chromatography in detecting real pepper samples. Thus, two CLIAs based on the nanobody and MPs were demonstrated as reliable tools for monitoring OTA in pepper., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The emergence of anti-drug antibodies (ADAs) poses significant impacts on the bioactivity and toxicity of biotherapeutics including proteins and peptides. Developing reliable assays to monitor the magnitudes of ADAs in blood samples is therefore considered a crucial task in animal and human studies throughout the development of biotherapeutics. Peptides represent a significant and fast-growing category of biotherapeutics for the management of a variety of indications. While peptides generally exhibit lower immunogenicity risks compared to biologics of larger sizes, drug developers are still required to conduct the risk-based immunogenicity assessment as mandated by the regulatory authorities. To address the need for efficient detection of ADAs against therapeutic peptides, we established a straightforward electrochemiluminescence immunoassay (ECLIA) based on direct binding strategy. Our assay demonstrates its applicability across various peptide therapeutics including marketed drugs and internal investigational compounds. Through stepwise tuning of the assay procedure, we identified several key factors such as buffer, detection reagent, plate type, and conjugation strategy that collectively contribute to the assay performance. Depending on the drug molecule and positive control antibody, the assay can achieve low single-digit to two-digit ng/ml sensitivity and ideal drug tolerance. In conclusion, this ECLIA platform presents a valuable and generic tool to expedite the development and validation of ADA assays for peptide-based therapeutics., Competing Interests: Declaration of Competing Interest All authors are currently full-time employee and could be shareholders of Takeda Pharmaceutical Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Conventional methods for diagnosing tuberculosis (TB), a significant global health challenge, often have drawbacks like time-consuming procedures, limited sensitivity, and the need for complex, expensive infrastructure. Hence, the development of electrochemical immunosensors has emerged as a promising strategy for TB detection due to their simplicity, speed, sensitivity, portability, and cost-effectiveness. In this study, we developed a rapid, simple, and low-cost immunosensor using a lab-made screen-printed electrode (SPE) based on the peptide TB 68-G as a recognition site. This synthetic peptide is composed of two important parts, one with an affinity for graphene materials and the other able to interact with anti-M. tuberculosis antibodies. This structural configuration allows for effective modification of the electrode surface while maintaining the ability to recognize the target. The proposed label-free electrochemical immunosensor was tested against M. tuberculosis antibodies and demonstrated a detection limit of 192 ng mL -1 with an R 2 value of 0.98. The diagnostic platform exhibited selectivity against nonspecific antibodies and successfully differentiated between negative and positive human serum samples with a 95 % confidence interval. This simple and affordable immunosensor holds great potential to impact TB control by enabling effective detection and improving disease surveillance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Several barriers for the development of an HCV vaccine still exist, including the genetic diversity of the virus, and the shortage of assessable models for in vitro and in vivo assays. Therefore, in this study, HCV epitope mapping was performed for 59 polyprotein sequences from 7 HCV genotypes. Around 2,880 peptides were considered epitopes for CD8 + T cells. The peptide induction of cytokines from Th1 and/or Th2 axes of the cellular immune response was assessed, indicating a tendency for Th2 axis. In vitro evaluation was performed using peptide microarray and a recombinant HLA-A*02:01 molecule. A total of 615 peptides of high reactivity to HLA-A*02:01 were identified, with predominance of leucine and tryptophan residues, highlighting their importance for TCR-epitope binding and CD8 + T activation. Finally, HCV-derived peptide patterns restricted to HLA-A2*02:01 observed in this study provide important information for the development of a multi-epitope-based pan-genotypic vaccine against the virus., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The impact of chirality on immune response has attracted great interest in cancer vaccine research recently. However, the study of chiral synthetic polypeptide hydrogels as cancer vaccines as well as of the impact of biomaterials themselves for antitumor immunotherapy has rarely been reported. Here, we show the key role of residue chirality of polypeptide hydrogels in antitumor immunity and local immune microenvironment regulation. Compared to poly(γ-ethyl-L-glutamate)-based hydrogels (L-Gel), poly(γ-ethyl-D-glutamate)-based hydrogels (D-Gel) induces enhanced level of immune cell infiltration. However, D-Gel causes higher levels of suppressive markers on antigen-presenting cells and even induces stronger T cell exhaustion than L-Gel. Finally, D-Gel establishes a local chronic inflammatory and immunosuppressive microenvironment and shows insufficient anti-tumor effects. Conversely, the milder host immune responses induced by L-Gel leads to more effective tumor inhibition. This study provides insights on the role of residue chirality in the regulation of local immune microenvironment and affecting antitumor immune response., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The inherent antigen cross-reactivity of the T cell receptor (TCR) is balanced by high specificity. Surprisingly, TCR specificity often manifests in ways not easily interpreted from static structures. Here we show that TCR discrimination between an HLA-A*03:01 (HLA-A3)-restricted public neoantigen and its wild-type (WT) counterpart emerges from distinct motions within the HLA-A3 peptide binding groove that vary with the identity of the peptide's first primary anchor. These motions create a dynamic gate that, in the presence of the WT peptide, impedes a large conformational change required for TCR binding. The neoantigen is insusceptible to this limiting dynamic, and, with the gate open, upon TCR binding the central tryptophan can transit underneath the peptide backbone to the opposing side of the HLA-A3 peptide binding groove. Our findings thus reveal a novel mechanism driving TCR specificity for a cancer neoantigen that is rooted in the dynamic and allosteric nature of peptide/MHC-I binding grooves, with implications for resolving long-standing and often confounding questions about T cell specificity., Competing Interests: Competing interests: C.A.K. and S.S.C. are inventors of patents related to the T cell receptor (TCR) sequences featured in this manuscript and are recipients of licensing revenue from Intima Bioscience shared according to Memorial Sloan Kettering Cancer Center (MSKCC) institutional policies. C.A.K. has consulted for or is on the scientific advisory boards for Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma, and T-knife, and is a scientific co-founder and equity holder in Affini-T Therapeutics. S.S.C. is a scientific advisor and equity holder in Affini-T Therapeutics. B.M.B. is an inventor on patents relating to differences between mutant and self in identifying immunogenic neoantigens has consulted for or received funding from Merck, Pfizer, Eureka Therapeutics, and EnaraBio, and is on the scientific advisory board of T-cure Bioscience. The other authors declare no competing interests., (© 2025. The Author(s).)

Background: T cell receptor (TCR)-engineered T cells targeting neoantigens originated from mutations in KRAS gene have demonstrated promising outcomes in clinical trials against solid tumors. However, the challenge lies in developing tumor-specific TCRs that avoid cross-reactivity with self-antigens to minimize the possibility of severe clinical toxicities. Current research efforts have been put towards strategies to eliminate TCR off-target recognition., Methods: Naive T cell repertoire was used for screening KRAS G12D -reactive TCRs. Specific TCRs were subsequently identified and their functionality was assessed using TCR Jurkat cells and TCR T cells. Peptide specificity was evaluated using the X-scan assay. To enhance TCR specificity for KRAS G12D and reduce their reactivity to self-peptide SMC1A 29-38 , mammalian TCR display libraries were employed for the design of modification in the complementarity-determining region (CDR)., Results: HLA-A*11:01-restricted TCRs targeting the KRAS G12D epitope were isolated, and TCR1 was characterized with superior functional avidity and specificity. Alongside a robust recognition of endogenous KRAS G12D epitope, this TCR displayed cross-reactivity with the SMC1A 29-38 epitope. With an approach utilizing structural-guided mutations in the CDR-1A region of TCR1, we obtained an engineered TCR variant (TCR1a7). Functional characterization of TCR1a7 showed that this TCR not only exhibited enhanced specificity towards KRAS G12D , but also demonstrated successful elimination of the off-target recognition of SMC1A 29-38 ., Conclusions: TCRs targeting the KRAS G12D peptide could be isolated from naive T cell repertoires. Integrating the TCR-peptide-HLA complex structure with a mammalian TCR library system could serve as a functional strategy to reduce potential TCR cross-reactivity with self-antigens, such as SMC1A 29-38 . Our findings evidenced an operable method to enhance TCR peptide specificity, while maintaining advanced functional avidity and potent anti-tumor activity., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University ( the approval number: 2023–234). All individuals signed an informed consent form. Consent for publication: Not applicable. Competing interests: Patent has been filed for KRASG12D specific TCRs presented here. All other authors declare no competing interests., (© 2025. The Author(s).)

T cells recognize peptides displayed on the surface of cells on MHC molecules. Genetic variation in MHC genes alters their peptide-binding repertoire and thus influences the potential immune response generated against pathogens. Both gorillas and chimpanzees show reduced diversity at their MHC class I A (MHC-A) locus compared to humans, which has been suggested to be the result of a pathogen-mediated selective sweep. More specifically, gorillas lack A3 lineage alleles while chimpanzees seem to have lost the A2 lineage. While previous studies showed this using phylogenetic analysis, here, we take an in silico functional approach and use the peptide-MHC binding prediction software NetMHCpan to examine the peptide-binding repertoires of common human, chimpanzee, and gorilla MHC-A molecules. We find that both gorillas and chimpanzees lack the A02 peptide binding specificity (supertype) despite gorillas being expected to have this specificity since they kept the A2 lineage. Additionally, we show that human MHC molecules with the A02 specificity bind fewer virus-derived peptides than other MHC molecules. We also do not find differential presentation of self-peptides by the A02 supertype, making the purpose of maintaining this specificity in high frequencies in the human population unclear. Taken together, we hypothesize that poor presentation of viral peptides by A02 supertype MHC molecules could have resulted in a selective sweep in chimpanzees and/or gorillas, though we could not identify a specific virus that may have caused this sweep., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Interferon (IFN)-α is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but the effect of IFN-α on the antigen repertoire of HLA Class I (HLA-I) in pancreatic β-cells is unknown. Here we characterize the HLA-I antigen presentation in resting and IFN-α-exposed β-cells and find that IFN-α increases HLA-I expression and expands peptide repertoire to those derived from alternative mRNA splicing, protein cis-splicing and post-translational modifications. While the resting β-cell immunopeptidome is dominated by HLA-A-restricted peptides, IFN-α largely favors HLA-B and only marginally upregulates HLA-A, translating into increased HLA-B-restricted peptide presentation and activation of HLA-B-restricted CD8 + T cells. Lastly, islets of patients with T1D show preferential HLA-B hyper-expression when compared with non-diabetic donors, and islet-infiltrating CD8 + T cells reactive to HLA-B-restricted granule peptides are found in T1D donors. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward alternative epitopes presented by HLA-B, hence recruiting T cells with a distinct repertoire that may be relevant to T1D pathogenesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Class I major histocompatibility complex (MHC-I) proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. The chaperones tapasin and TAPBPR promote the selection of immunogenic antigens from a large pool of intracellular peptides. Interactions of chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Here, we leverage a high-fidelity TAPBPR variant and conformationally stabilized MHC-I, to determine the solution structure of the human antigen editing complex bound to a peptide decoy by cryogenic electron microscopy (cryo-EM) at an average resolution of 3.0 Å. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the P2/P3 peptide anchors, where conserved MHC-I residues stabilize incoming peptides through backbone-focused contacts. Finally, using our high-fidelity chaperone, we demonstrate robust peptide exchange on the cell surface across multiple clinically relevant human MHC-I allomorphs. Our work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications., Competing Interests: Competing interests statement:N.G.S. is listed as an inventor in a patent application related to this work.

Background: The immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential., Methods: To meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification., Results: We employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition (MAPKi) and to investigate alterations associated with early cellular responses to inhibitor treatment and acquired resistance to MAPKi. Our analyses demonstrated significant changes in the immunopeptidome early during MAPKi treatment and in the resistant state. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes that might represent exploitable targets for cancer immunotherapy., Conclusions: We have developed a new mass spectrometric approach that allowed us to investigate the effects of common MAPK inhibitors on the immunopeptidome of melanoma cells. This finally led to the discovery of new potential targets for cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Bernhardt, Rech, Berthold, Lappe, Herbel, Erhard, Paschen, Schilling and Schlosser.)

Recognition of antigens by T cell receptors (TCRs) is a key component of adaptive immunity. Understanding the structures of these TCR interactions provides major insights into immune protection and diseases, and enables design of therapeutics, vaccines and predictive modeling algorithms. Previously, we released TCR3d, a database and resource for structures of TCRs and their recognition. Due to the growth of available structures and categories of complexes, the content of TCR3d has expanded substantially in the past 5 years. This expansion includes new tables dedicated to TCR mimic antibody complex structures, TCR-CD3 complexes and annotated Class I and II peptide-MHC complexes. Additionally, tools are available for users to calculate docking geometries for input TCR and TCR mimic complex structures. The core tables of TCR-peptide-MHC complexes have grown by 50%, and include binding affinity data for experimentally determined structures. These major content and feature updates enhance TCR3d as a resource for immunology, therapeutics and structural biology research, and enable advanced approaches for predictive TCR modeling and design. TCR3d is available at: https://tcr3d.ibbr.umd.edu., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Severe COVID-19 can trigger a cytokine storm, leading to acute respiratory distress syndrome (ARDS) with similarities to superantigen-induced toxic shock syndrome. An outstanding question is whether SARS-CoV-2 protein sequences can directly induce inflammatory responses. In this study, we identify a region in the SARS-CoV-2 S2 spike protein with sequence homology to bacterial super-antigens (termed P3). Computational modeling predicts P3 binding to sites on MHC class I/II and the TCR that partially overlap with sites for the binding of staphylococcal enterotoxins B and H. Like SEB and SEH derived peptides, P3 stimulated 25-40% of human CD4+ and CD8 + T-cells, increasing IFN-γ and granzyme B production. viSNE and SPADE profiling identified overlapping and distinct IFN-γ+ and GZMB+ subsets. The super-antigenic properties of P3 were further evident by its selective expansion of T-cells expressing specific TCR Vα and Vβ chain repertoires. In vivo experiments in mice revealed that the administration of P3 led to a significant upregulation of proinflammatory cytokines IL-1β, IL-6, and TNF-α. While the clinical significance of P3 in COVID-19 remains unclear, its homology to other mammalian proteins suggests a potential role for this peptide family in human inflammation and autoimmunity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

αβ T cell receptor (TCR) recognition of peptide-MHC complexes lies at the core of adaptive immunity, balancing specificity and cross-reactivity to facilitate effective antigen discrimination. Early structural studies established basic frameworks helpful for understanding and contextualizing TCR recognition and features such as peptide specificity and MHC restriction. However, the growing TCR structural database and studies launched from structural work continue to reveal exceptions to common assumptions and simplifications derived from earlier work. Here we explore our evolving understanding of TCR recognition, illustrating how structural and biophysical investigations regularly uncover complex phenomena that push against paradigms and expand our understanding of how TCRs bind to and discriminate between peptide/MHC complexes. We discuss the implications of these findings for basic, translational, and predictive immunology, including the challenges in accounting for the inherent adaptability, flexibility, and occasional biophysical sloppiness that characterize TCR recognition., (© 2025 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A novel panel of peptide for serological identification of Borrelia burgdoferi sensu stricto, Borrelia garinii and Borrelia afzelii was developed and assessed in this study. The diagnostic algorithm of the novel test was initially trained testing 10 US human sera including 3 early-stage and 3 late-stage Lyme disease positive sera, 2 sera positive for Babesia and 2 sera positive for Syphilis, all purchased from a private biorepository. Findings were then corroborated testing (a) 33 additional EU follow-up positive sera from seroconverted patients bitten by ticks that tested positive for B. burgdorferi sensu stricto (No 2), Borrelia garinii (No 14), Borrelia afzelii (No 15) Borrelia valaisiana (No 2), and (b) 40 negative sera from US healthy donors. Results of preliminary US sera testing showed successful detection of IgM and IgG antibodies and correct identification of Borrelia burgdorferi sensu stricto in all the samples tested. Analysis of EU follow-up sera showed much higher sensitivity and accuracy when IgM and IgG were tested combined together rather than separately. Sensitivity and accuracy in species identification of the anti-IgM + IgG multiplex peptide ELISA was 93.5 % and 96.5 % respectively; lower test performance was observed when IgM (i.e. sensitivity = 58.1 %; correct identification = 88.8 %) and IgG testing (i.e. sensitivity = 74.1 %; correct identification = 96.5 %) were carried out separately. Overall specificity of the anti-IgM, anti-IgG and anti-IgM + IgG multiplex peptide ELISA calculated on a total number of 46 negative sera included in this study was 91.3 %, 95.6 and 93.4 %, respectively., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Bovine IgG1 Fc receptor (boFcγRI) is a homologue to human FcγRI (CD64) that has three extracellular Ig-like domains and can bind bovine IgG1 with high affinity. Identification of the linear epitope for Fc-binding on boFcγRI provides new insights for the IgG-Fcγ interaction and FcγR-targeting drugs development., Methods: The boFcγRI molecules were expressed on cell surface of the boFcγRI -transfected COS-7 cells. The extracellular domain of boFcγRI was expressed in Escherichia coli (E. coli) BL21, and the soluble boFcγRI was purified by Ni-chelation chromatography followed by refolding. To identify the Fc-binding epitope on the boFcγRI, peptides derived from the membrane-distal extracellular domain (EC2) of boFcγRI were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin (BSA). Binding of bovine IgG1 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal IgG-binding was further modified by truncation and mutation. The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay, respectively., Results: The minimal effective peptide TNLSHNGI corresponding to the sequence 142-149 of boFcγRI was found to bind bovine IgG1 specifically in Dot-blot suggesting it represents a linear ligand-binding epitope located in the putative E-F loop of the EC2 domain on the receptor. Mutation analysis of the peptide showed that the residues of Thr 142 , Asn 143 , Leu 144 , Gly 148 and Ile 149 within the linear epitope are critical for IgG1-binding. The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRII with IC 50 of 20.27 μmol/L, and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRI transfected cells with IC 50 of 86.75 μmol/L., Conclusions: The linear epitope for Fc-binding as well as its crucial residues of EC2 domain on boFcγRI was identified using synthetic peptides. The Fc-binding peptide showed well capability of regulating boFcγRI-IgG1 interaction on cell surface., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest., (Copyright © 2025 Elsevier B.V. All rights reserved.)

The anaphylatoxins C3a and C5a are products of the complement cascade that play important and interrelated roles in health and disease. Both are potential targets for anti-inflammatory active immunotherapies in which a patient's own immune system is stimulated to produce therapeutic immune responses against problematic self-molecules. However, the complex and time-dependent interrelations between the two molecules make dual targeting challenging. To investigate a dual-target active immunotherapy against C3a and C5a and to systematically study the effect of varied degrees of responses against both targets, the study employed self-assembled peptide immunogens capable of displaying a broad range of epitope compositions and Design-of-Experiments (DoE) approaches. Peptide nanofibers contained B-cell epitopes of C3a and C5a in defined quantities, and intranasal immunization raised systemic and mucosal immunity against each target. In a lipopolysaccharide-induced model of sepsis, increasing anti-C5a responses are protective, whereas increasing anti-C3a responses are detrimental, and survival rates are negatively correlated with anti-C3a/anti-C5a IgG titer ratio. This work highlights the interplay between the two molecules by making use of a modular, defined, and easily adjusted biomaterial-based active immunotherapy platform., (© 2024 Wiley‐VCH GmbH.)

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Dual Opdivo plus Yervoy immunotherapy, targeting the immune checkpoints PD1 and CTLA-4, is successful in clinical use. However, it is associated with a high incidence of adverse events, and its therapeutic efficacy needs improving. In this study, polymeric multivalent Fc-binding peptides (PLG-Fc-III-4C) are employed to fabricate a bispecific antibody (PD1/CTLA-4 BsAb) to potentiate dual immunotherapy targeting PD1 and CTLA-4. The PD1/CTLA-4 BsAb is prepared by mixing PLG-Fc-III-4C with aPD1 and aCTLA-4 in an aqueous solution for 3 h using the clinically optimal 3:1 proportion of aPD1 to aCTLA-4. PD1/CTLA-4 BsAb significantly inhibits tumors in MC38 colon cancer-bearing mice more effectively than the combination of aPD1 and aCTLA-4, with tumor suppression rates of 96.8% and 77.3%, respectively. It also induces a higher percentage of CD8 + T cells and increases the secretion of effector cytokines while reducing Treg levels in tumors compared to phosphate-buffered saline, indicating significant tumor immunity regulation. Mechanistically, a 6.3-fold increase in PD1/CTLA-4 BsAb accumulation in tumors due to the tumor targeting ability of aPD1, and the PD1/CTLA-4 BsAb significantly reduces the adverse colitis event in healthy mice, compared to aPD1 and aCTLA-4. Thus, these findings provide a novel approach to enhance antitumor therapy using aPD1 and aCTLA-4., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Leptospira , the pathogenic helical spirochetes that cause leptospirosis, is an emerging zoonotic disease with effective dissemination tactics in the host and can infect humans and animals with moderate or severe illnesses. Thus, peptide-based vaccines may be the most effective strategy to manage the immune response against Leptospira to close these gaps. In the current investigation, highly immunogenic proteins from the proteome of Leptospira interorgan serogroup Icterohaemorrhagie serovar Lai strain 56601 were identified using immunoinformatic methods. It was discovered that the conserved and most immunogenic outer membrane Lepin protein was both antigenic and non-allergenic by testing 15 linear B-cells and the ten best T-cell (Helper-lymphocyte (HTL) with the most significant number of HLA-DR binding alleles and the eight cytotoxic T lymphocyte (CTL)) epitopes. Furthermore, a 3D structural model of CTL epitopes was created using the Pep-Fold3 platform. Using the Autodock 4.2 docking server, research was conducted to determine how well the top-ranked CTL peptide models attach to HLA-A*0201 (PDB ID: 4U6Y). With HLA-A*0201, the epitope SSGTGNLHV binds with a binding energy of -1.29 kcal/mol. Utilizing molecular dynamics modeling, the projected epitope-allele docked complex structure was optimized, and the stability of the complex system was assessed. Therefore, this epitope can trigger an immunological response and produce effective Leptospira vaccine candidates. Overall, this study offers a unique vaccination candidate and may encourage additional research into leptospirosis vaccines.Communicated by Ramaswamy H. Sarma.

HLA-E is a nonclassical, nonpolymorphic, class Ib HLA molecule. Its primary function is to present a conserved nonamer peptide, termed VL9, derived from the signal sequence of classical MHC molecules to the NKG2x-CD94 receptors on NK cells and a subset of T lymphocytes. These receptors regulate the function of NK cells, and the importance of this role, which is conserved across mammalian species, probably accounts for the lack of genetic polymorphism. A second minor function is to present other, weaker binding, pathogen-derived peptides to T lymphocytes. Most of these peptides bind suboptimally to HLA-E, but this binding appears to be enabled by the relative stability of peptide-free, but receptive, HLA-E-β2m complexes. This, in turn, may favor nonclassical antigen processing that may be associated with bacteria infected cells. This review explores how the structure of HLA-E, bound to different peptides and then to NKG2-CD94 or T-cell receptors, relates to HLA-E cell biology and immunology. A detailed understanding of this molecule could open up opportunities for development of universal T-cell and NK-cell-based immunotherapies., (© 2025 The Author(s). Immunological Reviews published by John Wiley & Sons Ltd.)

Fungal infections are becoming an increasingly serious challenge in clinic due to the increase in drug resistance and the lack of anti-fungal drugs. Vaccination is a useful approach to prevent fungal infections. However, the balance between effectiveness and side effects presents a challenge in vaccine development. In this work, we designed a plant virus-based conjugate vaccine. The non-infectiveness and innate immunogenicity of plant viruses make this vaccine both safe and effective. By conjugating a fungal antigenic peptide to the tobacco mosaic virus (TMV), the resultant vaccine improved the uptake efficiency of antigenic peptides by antigen-presenting cells and enhanced the ability to target lymph nodes. The results of in vivo vaccination in mice showed a significant increase of antigen-specific IgG antibody levels induced by the TMV conjugate vaccine. This work suggests that TMV conjugate vaccines may become a potential vaccine candidate for preventing fungal infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Encrypted peptides (EPs) have been recently described as a new class of antimicrobial molecules. They have been found in numerous organisms and have been proposed to have a role in host immunity and as alternatives to conventional antibiotics. Intriguingly, many of these EPs are found embedded in proteins unrelated to the immune system, suggesting that immunological responses extend beyond traditional host immunity proteins. To test this idea, we synthesized and analyzed representative peptides derived from non-immune human proteins for their ability to exert antimicrobial and immunomodulatory properties. Most of the tested peptides from non-immune proteins, derived from structural proteins as well as proteins from the nervous and visual systems, displayed potent in vitro antimicrobial activity. These molecules killed bacterial pathogens by targeting their membrane, and those originating from the same region of the body exhibited synergistic effects when combined. Beyond their antimicrobial properties, nearly 90% of the peptides tested exhibited immunomodulatory effects, modulating inflammatory mediators, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). Moreover, eight of the peptides identified, collagenin-3 and 4, zipperin-1 and 2, and immunosin-2, 3, 12, and 13, displayed anti-infective efficacy in two different preclinical mouse models, reducing bacterial infections by up to four orders of magnitude. Altogether, our results support the hypothesis that peptides from non-immune proteins may have a role in host immunity. These results potentially expand our notion of the immune system to include previously unrecognized proteins and peptides that may be activated upon infection to confer protection to the host., Competing Interests: Declaration of interests C.F-N. provides consulting services to Invaio Sciences and is a member of the Scientific Advisory Boards of Nowture S.L. and Phare Bio. C.F-N. has received research funding or in-kind donations from United Therapeutics, Strata Manufacturing PJSC, and Procter & Gamble, none of which were used in support of this work. An invention disclosure associated with this work has been filed., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mucosal immune responses to vaccination are essential for achieving full protection against pathogens entering their host at mucosal sites. However, traditional parenteral immunization routes commonly fail to raise significant mucosal immunity. Sublingual immunization is a promising alternative delivery route to raise robust immune responses both systemically and at mucosal sites, and nanomaterial-based subunit vaccine platforms offer opportunities for raising epitope-specific responses. Here, sublingual immunization is reported using the Coil29 platform of coiled-coil self-assembling peptide nanofibers. The successful immunization with epitopes of varying physicochemical properties by including mucus-modulating components - namely sequences of proline, alanine, and serine (PAS) is demonstrated. PASylation is shown to decrease mucin complexation and increase epithelial penetration in vitro, enabling sublingual immunization against a variety of selected peptide epitopes in vivo. Coil29 fibers are also readily formed into tablets for solid-state dosing formulations and maintain their immunogenicity in this state. Previous sublingual peptide nanofiber immunotherapies have been based on different structures, such as highly stable β-sheets. The present work demonstrates that alternatively folded structures such as α-helical nanofibers can also be rendered sublingually immunogenic, enabling immunization with a variety of peptide epitopes and offering additional ways to specify mucus interactions, delivery state, dosing, and formulation., (© 2024 Wiley‐VCH GmbH.)

Despite the presence of strategically positioned anatomical barriers designed to protect the central nervous system (CNS), it is not entirely isolated from the immune system 1,2 . In fact, it remains physically connected to, and can be influenced by, the peripheral immune system 1 . How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding question. Here, in searching for molecular cues that derive from the CNS and enable its direct communication with the immune system, we identified an endogenous repertoire of CNS-derived regulatory self-peptides presented on major histocompatibility complex class II (MHC-II) molecules in the CNS and at its borders. During homeostasis, these regulatory self-peptides were found to be bound to MHC-II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. However, in neuroinflammatory disease, the presentation of regulatory self-peptides diminished. After boosting the presentation of these regulatory self-peptides, a population of suppressor CD4 + T cells was expanded, controlling CNS autoimmunity in a CTLA-4- and TGFβ-dependent manner. CNS-derived regulatory self-peptides may be the molecular key to ensuring a continuous dialogue between the CNS and the immune system while balancing overt autoreactivity. This sheds light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases., Competing Interests: Competing interests: M.W.K. and J.K. hold provisional patent applications related to findings presented here., (© 2024. The Author(s).)

Background: Gut commensal microbiota has been identified as a potential environmental risk factor for multiple sclerosis (MS), and numerous studies have linked the commensal microorganism with the onset of MS. However, little is known about the mechanisms underlying the gut microbiome and host-immune system interaction., Methods: We employed bioinformatics methodologies to identify human microbial-derived peptides by analyzing their similarity to the MHC II-TCR binding patterns of self-antigens. Subsequently, we conducted a range of in vitro and in vivo assays to assess the encephalitogenic potential of these microbial-derived peptides., Findings: We analyzed 304,246 human microbiome genomes and 103 metagenomes collected from the MS cohort and identified 731 nonredundant analogs of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55 ). Of note, half of these analogs could bind to MHC II and interact with TCR through structural modeling of the interaction using fine-tuned AlphaFold. Among the 8 selected peptides, the peptide (P3) shows the ability to activate MOG 35-55 -specific CD4 + T cells in vitro. Furthermore, P3 shows encephalitogenic capacity and has the potential to induce EAE in some animals. Notably, mice immunized with a combination of P3 and MOG 35-55 develop severe EAE. Additionally, dendritic cells could process and present P3 to MOG 35-55 -specific CD4 + T cells and activate these cells., Interpretation: Our data suggests the potential involvement of a MOG 35-55 -mimic peptide derived from the gut microbiota as a molecular trigger of EAE pathogenesis. Our findings offer direct evidence of how microbes can initiate the development of EAE, suggesting a potential explanation for the correlation between certain gut microorganisms and MS prevalence., Funding: National Natural Science Foundation of China (82371350 to GY)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The accumulation of senescent cells can lead to tissue degeneration, chronic inflammatory disease and age-related tumorigenesis. Interventions such as senolytics are currently limited by off-target toxicity, which could be circumvented by instead enhancing immune-mediated senescent cell clearance; however, immune surveillance of senescent cells is often impeded by immunosuppressive factors in the inflammatory microenvironment. Here, we employ a chimeric peptide as a 'matchmaker' to bind to the urokinase-type plasminogen activator receptor, a cell surface marker of senescent cells. This peptide modifies the cell surface with polyglutamic acid, promoting immune cell-mediated responses through glutamate recognition. By enhancing the recruitment of immune cells and directly coupling senescent cells and immune cells, we show that this chimeric peptide induces immune clearance of senescent cells and restores tissue homeostasis in conditions such as liver fibrosis, lung injury, cancer and natural aging in mice. This chimeric peptide introduces an immunological conversion strategy that rebalances the senescent immune microenvironment, offering a promising direction for aging immunotherapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ovarian cancer is generally diagnosed at late stages. Monoclonal antibodies (MAbs) targeting antigens in ovarian cancer are used in the clinic. Anti-Müllerian hormone receptor type 2 (AMHR2) is a receptor highly expressed in ovarian cancer and it is a potential target antigen for immunotherapy. Extracellular domain of AMHR2 was analysed in terms of 3D structure and physicochemical properties, and 3 peptide sequences (Peptides 1, 7 and 11) were determined as targets. MAb production protocol was performed, and 6 MAb clones showing high affinity for peptides were obtained. P3B1, P10A10, P10B6 and P2A6 clones were for peptide 11 (P11), P2C9 was for P7, and P6C5 was for P1. Antibody isotype of P2A6 was IgG2a and the others were of IgG1 isotype. MAb binding to the native recombinant protein (AMHR2-Fc) was analysed by enzyme-linked immunosorbent assay (ELISA) and MAb binding to AMHR2 expressed by SKOV-3 ovarian cancer cells was analysed by western blot and immunofluorescent staining. P3B1 showed strong, P10A10, P10B6 and P2C9 showed medium affinity for the native protein (AMHR2-Fc). P3B1 and P2C9 showed strong binding in western blot analysis. Clones showed moderate binding in immunoflorescent staining. A complement dependent cytotoxicity (CDC) experiment was conducted using MAbs and transfected SKOV-3 cells. P3B1 induced a significant CDC. Variable regions of P3B1 MAb were sequenced. In conclusion, MAbs for three different regions of AMHR2 were produced. One clone was shown to induce cytotoxicity in ovarian cancer cells and its sequence was determined for future use as a humanised therapeutic MAb., (© 2024 The Scandinavian Foundation for Immunology.)

Uronium peptide coupling agents (HBTU, HATU, and HCTU) create a special hazard as they are immune sensitizers. Few reported cases are mentioned in the literature; despite that, it is important to raise the awareness on the subject and to highlight the risk and potential symptoms that could occur to those who directly work in contact with uronium peptide coupling agents, as well as to the safety deputies in the universities and industries. Based on a personal experience, the health impact of laboratory exposure to HBTU is described, and the insights gained from the experience are developed. A skin irritation reaction and allergy symptoms induced by HBTU exposure are shown here as well as the rate of worsening of symptoms since the first allergic reaction. Recommendations for handling coupling agents more safely in the research laboratory will also be given, and a casuistry of the matter to help other lab-users to recognize, assess, minimize, prepare for emergencies (RAMP) process., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Toxoplasma gondii infects approximately 30% of the population, and there is currently no approved vaccine. Identifying immunogenic peptides with high affinity to different HLA molecules is a promising vaccine strategy. This study used an in silico approach using artificial neural networks to identify T. gondii peptides restricted to HLA-A*02, HLA-A*24, and HLA-B*35 alleles. Proteomes from seven T. gondii strains and transcriptomic data of overexpressed genes from T. gondii-RH in human PBMC were also used. Parasite protein sequences were analyzed with R 'Epitope Prediction' library. Peptide candidates were evaluated in the artificial neural networks based on the probabilities of output neurons (p > 0.5). The IFN-γ responses in PBMC from T. gondii seronegative and seropositive individuals were evaluated by ELISpot. Peptides with higher IFN-γ induction were evaluated to identify cytotoxic response in CD8 + T cells (CD107a). In silico analysis identified 36 peptides from T. gondii proteins with predicted affinity to HLA-A*02, A*24, and B*35 alleles. Experiments with PBMCs revealed that a peptide restricted to HLA-A02 (P1: FLFAWITYV) induced a significant increase in IFN-γ-producing cells (p = 0.004). For HLA-A24, a peptide (P8: VFAFAFAFFLI) also induced a significant IFN-γ response (p = 0.004), while for the HLA-B*35 allele, the P6 peptide (YPIAPSFAM) induced a response that differed significantly from the control (p = 0.05). These peptides induced also a significant percentage of central memory CD8 + T cells expressing the degranulation marker CD107a (p < 0.05). Finally, we identified three T. gondii peptides that induced IFN-γ response, and a cytotoxic response measured by CD107a expression on CD45RAneg-CD8 cells. These peptides could be considered part of a multi-epitope vaccine against toxoplasmosis in humans., Competing Interests: Declarations. Ethical approval: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethics Committee of the Faculty of Health Sciences at the University of Quindio (Act Number 23. July 12 2019). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cancer remains a major global health challenge. Immunotherapy has revolutionized the management of cancer, yet only a limited number of patients respond to such treatments. This is largely attributed to the immunosuppressive tumor microenvironment, which diminishes the effectiveness of immunotherapy. Recent studies have underscored the potential of naturally derived caerin 1 peptides, particularly caerin 1.1 and caerin 1.9, which exhibit strong antitumor effects and enhance the efficacy of immunotherapies in animal models. This review encapsulates the current research aimed at augmenting the effectiveness of immunotherapy, focusing on the role of caerin 1.1 and caerin 1.9 in boosting immunotherapeutic outcomes, elucidating possible mechanisms, and discussing their limitations and challenges.

There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (Δ flhD Δ clbP ). Δ flhD Δ clbP -derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (Δ flhD -MVs). In addition, glycoengineered Δ flhD Δ clbP -MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14 + MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14 + MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g . an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of Δ flhD Δ clbP -MVs in the context of vaccine safety.

We have designed and produced 39 amino acid peptide mimics of the Torpedo and human acetylcholine receptors' (AChRs) main immunogenic regions (MIRs). These conformationally sensitive regions consist of three non-contiguous segments of the AChR α-subunits and are the target of 50-70% of the anti-AChR autoantibodies (Abs) in human myasthenic serum and in the serum of rats with a model of that disease, experimental autoimmune myasthenia gravis (EAMG), induced by immunizing the rats with the Torpedo electric organ AChR. These MIR segments covalently joined together bind a significant fraction of the monoclonal antibodies (mAbs) raised in rats against electric organ AChR. Many of these mAbs cross react with the rat neuromuscular AChR MIR and induce myasthenic symptoms when injected into naïve rats. The human MIR mimic peptide (H39MIR) is evolutionarily related to that of the Torpedo electric organ MIR mimic peptide ( T 39MIR) with eight amino acid differences between the two MIR mimics. The mAbs raised to the electric organ AChR MIR cross react with the human and scores of other species' neuromuscular AChRs. However, the mAbs do not cross react with the H39MIR mimic attached to the N-terminus of an intein-chitin-binding domain (H39MIR-IChBD) even though they do bind to the T 39MIR-IChBD construct. To account for this difference in binding anti-MIR mAbs, each of the eight human amino acids was substituted individually into the T 39MIR-IChBD, and four of them were found to weaken mAb recognition. Substituting the corresponding four Torpedo amino acids individually and in combination into the homologous positions in H39MIR-IChBD makes chimeric human MIR mimic peptides ( T /H39MIR), some of which bind anti-MIR mAbs and anti-MIR Abs from rat EAMG and human MG sera. The best mAb binding chimeric peptide constructs may potentially serve as the basis of a diagnostic anti-MIR Ab titer assay that is both prognostic and predictive of disease severity. Furthermore, the best peptides may also serve as the targeting element of a non-steroidal antigen-specific treatment of MG to remove anti-AChR MIR Abs, either as fused to the N-terminals of the human immunoglobin F c fragment or as the targeting component of a T cell chimeric autoantibody receptor (CAAR) directed to anti-MIR memory B cells for elimination.

Based on the success of cancer immunotherapy, personalized cancer vaccines have emerged as a leading oncology treatment. Antigen presentation on MHC class I (MHC-I) is crucial for the adaptive immune response to cancer cells, necessitating highly predictive computational methods to model this phenomenon. Here, we introduce HLApollo, a transformer-based model for peptide-MHC-I (pMHC-I) presentation prediction, leveraging the language of peptides, MHC, and source proteins. HLApollo provides end-to-end treatment of MHC-I sequences and deconvolution of multi-allelic data, using a negative-set switching strategy to mitigate misassigned negatives in unlabelled ligandome data. HLApollo shows a 12.65% increase in average precision (AP) on ligandome data and a 4.1% AP increase on immunogenicity test data compared to next-best models. Incorporating protein features from protein language models yields further gains and reduces the need for gene expression measurements. Guided by clinical use, we demonstrate pan-allelic generalization which effectively captures rare alleles in underrepresented ancestries., Competing Interests: Competing interests: The authors of this study are or were employees of Genentech Inc. at the time this work was done. The architecture of the deep learning method described here is related to a US patent filing (US-20220122690-A1) with K.L., N.L., W.J.T, Q.B., L.D., and S.J. as inventors., (© 2024. The Author(s).)

The discovery of tumor-derived neoantigens which elicit an immune response through major histocompatibility complex (MHC-I/II) binding has led to significant advancements in immunotherapy. While many neoantigens have been discovered through the identification of non-synonymous mutations, the rate of these is low in some cancers, including head and neck squamous cell carcinoma. Therefore, the identification of neoantigens through additional means, such as aberrant splicing, is necessary. To achieve this, we developed the splice isoform neoantigen evaluator (SINE) pipeline. Our tool documents peptides present on spliced or inserted genomic regions of interest using Patient Harmonic-mean Best Rank scores, calculating the MHC-I/II binding affinity across the complete human leukocyte antigen landscape. Here, we found 125 potentially immunogenic events and 9 principal binders in a cohort of head and neck cancer patients where the corresponding wild-type peptides display no MHC-I/II affinity. Further, in a melanoma cohort of patients treated with anti-PD1 therapy, the expression of immunogenic splicing events identified by SINE predicted response, potentially indicating the existence of immune editing in these tumors. Overall, we demonstrate SINE's ability to identify clinically relevant immunogenic neojunctions, thus acting as a useful tool for researchers seeking to understand the neoantigen landscape from aberrant splicing in cancer.

Background and Aims: In coeliac disease, the clinical role of the urinary gluten immunogenic peptide is unclear. It has been suggested it can be a non-invasive marker of villous atrophy. Therefore, we present the largest cross-sectional clinical data in patients with coeliac disease to establish the diagnostic accuracy of the urinary gluten immunogenic peptide in identifying villous atrophy., Methods: Patients providing urinary gluten immunogenic peptide were identified between September 2018 and August 2023 at the National Health Service (NHS) England National Centre for Non-Responsive and Refractory CD. In our retrospective study, the results of the urinary gluten immunogenic peptide test collected within 7 days, self-reported adherence to a gluten free diet reported within 3 months, serology collected within 7 days (immunoglobulin A - tissue transglutaminase and endomysial antibody) and histology at the time of endoscopy were compared in individuals with coeliac disease who were either asymptomatic and undergoing remission biopsies (group 1), non-responsive coeliac disease (group 2) and refractory coeliac disease on immunosuppressive therapy (group 3). Associations between dichotomous variables were calculated using chi-squared test., Results: In group 1 the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detecting villous atrophy were 42.9%, 83.3.%, 64.3% and 67.6% respectively. In group 2 the sensitivity, specificity, PPV and NPV for detecting villous atrophy were 36.2%, 79.0%, 39.5% and 76.6% respectively. In group 3 the sensitivity, specificity, PPV and NPV for detecting villous atrophy were 56.3%, 70.6%, 73.0% and 53.3%. More patients on immunosuppression had a positive urinary gluten immunogenic peptide than those not on immunosuppression (43.3% vs 24.1%, p<0.001)., Conclusions: The urinary gluten immunogenic peptide does not have a role in identifying villous atrophy. Therefore, to assess for villous atrophy an upper gastrointestinal endoscopy is still required.

SARS-CoV-2 is an enveloped virus. Proteins of the lipid based envelope are not rigidly arranged as compared to non-enveloped viruses lacking lipid based outer layer. Rigidly arranged multivalent display has been reported to be important for potent immune stimulation. We assembled himeric virus-like-particle (VLP) where the core of the particle is composed of the coat protein of Acinetobacter phage. Peptide-based antigen from receptor binding domain (RBD) of SARS-CoV-2 spike protein has been displayed on the coat based VLP matrix using spy-tag/spy-catcher split inteine conjugation system that allows spontaneous, irreversible isopeptide bond formation. Both the matrix as well as peptide have been purified from bacteria which is an easy platform for protein purification. We used the closed state of spike trimer for epitope mapping as this is the conformation of the spike that the immune system visualizes unlike the open state that appears when the virus tries to attach to receptor. Mice based immunization studies were used to test immunogenicity of the antigens. The work demonstrates that peptide-based antigens displayed in high densities can induce neutralizing antibody production unlike free peptide. Careful choice of peptides can deliver better candidates. Also, small size allows easy improvisation. Production in bacteria offers cheaper and robust purification option., Competing Interests: Declaration of competing interest Authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The interaction between peptides and major histocompatibility complex (MHC) molecules is pivotal in autoimmunity, pathogen recognition and tumor immunity. Recent advances in cancer immunotherapies demand for more accurate computational prediction of MHC-bound peptides. We address the generalizability challenge of MHC-bound peptide predictions, revealing limitations in current sequence-based approaches. Our structure-based methods leveraging geometric deep learning (GDL) demonstrate promising improvement in generalizability across unseen MHC alleles. Further, we tackle data efficiency by introducing a self-supervised learning approach on structures (3D-SSL). Without being exposed to any binding affinity data, our 3D-SSL outperforms sequence-based methods trained on ~90 times more data points. Finally, we demonstrate the resilience of structure-based GDL methods to biases in binding data on an Hepatitis B virus vaccine immunopeptidomics case study. This proof-of-concept study highlights structure-based methods' potential to enhance generalizability and data efficiency, with possible implications for data-intensive fields like T-cell receptor specificity predictions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Porcine bocavirus (PBoV), classified within the genus Bocaparvovirus, has been reported worldwide. PBoV has been divided into group 1, group 2, and group 3. PBoV group 3 (G3) viruses are the most prevalent in China. Currently, effective serological methods for the detection of antibodies against PBoV G3 are limited. In this study, we developed an indirect ELISA using a synthetic VP1 peptide designed on the basis of the conserved region of the PBoV VP1 protein as a coating antigen. Through matrix titration, the optimal coating concentration of the VP1 peptide (0.5 μg/mL), serum dilution (1:200), and working concentration of the secondary antibody (1:50,000) were determined. The cutoff value of this developed ELISA was set as 0.4239. Further investigations revealed that this developed ELISA had no cross-reactivity with positive serum antibodies against FMDV-O, FMDV-A, PRV, ASFV, SF, PCV2, PEDV, and TGEV. The detection limit of the method was a 1:1600 dilution of standard positive serum against PBoV G3. The coefficients of variation for both the intra- and interassay data were lower than 10%. A total of 1373 serum samples collected from 12 provinces in China between 2022 and 2023 were subjected to indirect ELISA. The results showed that 47.56% of the samples were PBoV G3 positive. These results reveal that peptide-based ELISA is a reliable and cost-effective method for detecting PBoV G3 antibodies. It also facilitates the investigation of the prevalence and distribution of PBoV G3.

Plant endogenous signaling peptides shape growth, development and adaptations to biotic and abiotic stress. Here, we identify C-TERMINALLY ENCODED PEPTIDEs (CEPs) as immune-modulatory phytocytokines in Arabidopsis thaliana. Our data reveals that CEPs induce immune outputs and are required to mount resistance against the leaf-infecting bacterial pathogen Pseudomonas syringae pv. tomato. We show that effective immunity requires CEP perception by tissue-specific CEP RECEPTOR 1 (CEPR1) and CEPR2. Moreover, we identify the related RECEPTOR-LIKE KINASE 7 (RLK7) as a CEP4-specific CEP receptor contributing to CEP-mediated immunity, suggesting a complex interplay of multiple CEP ligands and receptors in different tissues during biotic stress. CEPs have a known role in the regulation of root growth and systemic nitrogen (N)-demand signaling. We provide evidence that CEPs and their receptors promote immunity in an N status-dependent manner, suggesting a previously unknown molecular crosstalk between plant nutrition and cell surface immunity. We propose that CEPs and their receptors are central regulators for the adaptation of biotic stress responses to plant-available resources., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

T cell activity is governed by T cell receptor (TCR) signaling and constrained by immune checkpoint molecules, including programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and lymphocyte activation gene 3 (LAG-3). The basis for how LAG-3 binds to human leukocyte antigen class II molecules (HLA-II) remains unknown. Here, we present the 3.4-angstrom crystal structure of a LAG-3-peptide-HLA-II complex and probe the energetics of the complex interface. Coincident with the HLA-II binding site of the ancestrally related, monomeric CD4 receptor, the LAG-3 homodimer laterally engages two HLA-II molecules via distal D1 domain surfaces, imposing a 38° angular offset. The LAG-3-HLA-II interface is discontinuous and lacks involvement of the D1 extra loop, a binding site for anti-LAG-3 therapeutic monoclonal antibodies. Upon HLA-II binding, intrinsically mobile loops of the LAG-3 molecule become ordered, with contact residues highly conserved across HLA-DR, DQ, and DP allomorphs. Our data provide a structural foundation for development of immunomodulatory approaches targeting LAG-3.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID 50 ] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC 50 ] < 50 μg/mL) and total lineage-concentrations estimates of 50-200 μg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Many promising targets for adoptive T cell therapy (ACT) are self-antigens, but self-reactive T cells are generally eliminated during thymic selection or diverted to regulatory phenotypes. To bypass T cell tolerance and obtain potent and safe T cell therapeutics, we developed T-Switch, an in vitro T cell receptor (TCR) engineering platform for the creation, modification, and comprehensive profiling of TCRs that can target self-antigens. T-Switch first expands T cells that recognize a "foreign" peptide closely related to a self-antigen. The fine specificity of the TCR is then modified by directed evolution of the peptide binding region to switch its specificity to the self-antigen of interest. We applied T-Switch to engineer synthetic TCRs reactive to a tumor-associated self-antigen, validated the safety and efficacy of this approach, and detected no off-target recognition as measured against the human proteome. Thus, T-Switch represents a resource for the creation of collections of highly sensitive synthetic TCRs for T cell-based immunotherapies., Competing Interests: Declaration of interests N.S.A. and S.J.E. are inventors of and have submitted a patent on the T-Switch technology. T.K. and S.J.E. are also founders of and hold equity in TScan Therapeutics. S.J.E. is also founder of MAZE Therapeutics and Mirimus and serves on the scientific advisory board of TSCAN Therapeutics and MAZE Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Mutations commonly occur in cancer cells, arising neoantigen as potential targets for personalized immunotherapy of lung adenocarcinoma (LUAD). However, the substantial heterogeneity observed among individuals and distinct foci within the same patient presents significant challenges in formulating immunotherapy strategies. The aim of the work is to characterize the mutation pattern and identify neopeptides across different patients and diverse foci within the same patients with LUAD., Methods: Seven lung adenocarcinoma samples and matched tissues/blood are collected from 4 patients with LUAD for whole exome sequencing, mutation signature analysis, HLA binding prediction and neoantigen screening. Dimeric HLA-A2 molecules were prepared by Bac-to-Bac baculovirus expression system to establish a T cell stimulation system based on HLA-A2-coated artificial antigen-presenting cells for the validation of immunogenic neopeptides., Results: Similar mutation pattern with predominant missense mutation and high tumor mutation burden was observed across individuals with lung adenocarcinomas and between non-invasive and invasive foci. We screened and identified 3 consistent mutated genes among 100 top genes with highest mutation scores contributed across 4 patients, and 3 mutated peptides among 30 with highest HLA-A2 binding affinity distributed in at least 2 out of 4 foci in the same patient. Notably, LUAD-7-MT peptide encoded by NANOGNB demonstrated higher immunogenicity in promoting CD8+ T cells proliferation and IFN-γ secretion than the corresponding wildtype peptide., Conclusions: This study provides an in-depth analysis of mutation characteristics of LUAD and establishes a neoantigen screening and validation system for identifying immunogenicity neopeptide across individual patients and diverse foci in the same patient with multifocal LUAD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Jiang, Zhao, Wu, Xiong, Wu and Weng.)

Allogeneic stem cell transplantation (alloSCT) provides a curative treatment option for hematological malignancies. After HLA-matched alloSCT, donor-derived T cells recognize minor histocompatibility antigens (MiHAs), which are polymorphic peptides presented by HLA on patient cells. MiHAs are absent on donor cells due to genetic differences between patient and donor. T cells targeting broadly expressed MiHAs induce graft-versus-leukemia (GvL) reactivity as well as graft-versus-host disease (GvHD), while T cells for MiHAs with restricted or preferential expression on hematopoietic or non-hematopoietic cells may skew responses toward GvL or GvHD, respectively. Besides tissue expression, overall strength of GvL and GvHD is also determined by T-cell frequencies against MiHAs.Here, we explored the use of DNA barcode-labeled peptide-MHC multimers to detect and monitor antigen-specific T cells for the recently expanded repertoire of HLA-I-restricted MiHAs. In 16 patients who experienced an immune response after donor lymphocyte infusion, variable T-cell frequencies up to 30.5% of CD8 + T cells were measured for 49 MiHAs. High T-cell frequencies above 1% were measured in 12 patients for 19 MiHAs, with the majority directed against mismatched MiHAs, typically 6-8 weeks after donor lymphocyte infusion and at the onset of GvHD. The 12 patients included 9 of 10 patients with severe GvHD, 2 of 3 patients with limited GvHD and 1 of 3 patients without GvHD.In conclusion, we demonstrated that barcoded peptide-MHC multimers reliably detect and allow monitoring for MiHA-specific T cells during treatment to investigate the kinetics of immune responses and their impact on development of GvL and GvHD after HLA-matched alloSCT., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Acinetobacter baumannii, is among the highest priority bacteria according to the WHO categorization which necessitate the exploration of alternative strategies such as vaccination. OmpA, BamA, and Omp34 are assigned as appropriate antigens to serve in vaccine development against this pathogen. Experimentally validated exposed epitopes of OmpA and Omp34 along with selected exposed epitopes predicted by an integrative in silico approach were represented by the barrel domain of BamA as a scaffold. Among the 8 external loops of BamA, 5 loops were replaced with selected loops of OmpA and Omp34. The designed antigen was analyzed regarding the physicochemical properties, antigenicity, epitope retrieval, topology, structure, and safety. BamA is a two-domain OMP with a 16-stranded barrel in which L4, L6, and L7 were the longest loops of BamA in order. The designed antigen consisted of 478 amino acids with antigen probability of 0.7793. The novel antigen was a 16-stranded barrel. No identical 8-meric peptides were found in the human proteome against the designed antigen sequence. The designed construct was safe regarding the allergenicity, toxicity, and human proteome reactivity. The designed antigen could develop higher protection against A. baumannii in comparison to either OmpA, BamA, or Omp34 alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: T cells form one of the key pillars of adaptive immunity. Using their surface bound T cell antigen receptors (TCRs), these cells screen millions of antigens presented by major histocompatibility complex (MHC) or MHC-like molecules. In other protein families, the dynamics of protein-protein interactions have important implications for protein function. Case studies of TCR:class I peptide-MHCs (pMHC-Is) structures have reported mixed results on whether the binding interfaces undergo conformational change during engagement and no robust statistical quantification has been done to generalise these results. Thus, it remains an open question of whether movement occurs in the binding interface that enables the recognition and activation of T cells., Methods: In this work, we quantify the conformational changes in the TCR:pMHC-I binding interface by creating a dataset of 391 structures, comprising 22 TCRs, 19 MHC alleles, and 79 peptide structures in both unbound (apo) and bound (holo) conformations., Results: In support of some case studies, we demonstrate that all complementarity determining region (CDR) loops move to a certain extent but only CDR3α and CDR3β loops modify their shape when binding pMHC-Is. We also map the contacts between TCRs and pMHC-Is, generating a novel fingerprint of TCRs on MHC molecules and show that the CDR3α tends to bind the N-terminus of the peptide and the CDR3β tends to bind the C-terminus of the peptide. Finally, we show that the presented peptides can undergo conformational changes when engaged by TCRs, as has been reported in past literature, but novelly show these changes depend on how the peptides are anchored in the MHC binding groove., Conclusions: Our work has implications in understanding the behaviour of TCR:pMHC-I interactions and providing insights that can be used for modelling Tcell antigen specificity, an ongoing grand challenge in immunology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 McMaster, Thorpe, Rossjohn, Deane and Koohy.)

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/ L. reuteri , KC1-VP60/ L. reuteri , and MY-VP60/ L. reuteri . The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/ L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

To combat with emerging SARS-CoV-2 variants of concern (VOCs), we report the identification of a set of unique HWK-motif peptide ligands for the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein from a phage-displayed peptide library. These HWK-motif peptides exhibited nanomolar affinity for RBD. Among them, the peptide, HWKAVNWLKPWT (SP-HWK), had not only the highest affinities for RBD and trimer S protein, but also broad-spectrum affinities for RBDs from VOCs. Molecular dynamics simulations and competitive ELISA revealed a conserved pocket between the cryptic and the outer faces of RBD for SP-HWK binding, distinct from the human angiotensin-converting enzyme 2 receptor binding site. By coupling SP-HWK to agarose gel, the as-prepared affinity gel could efficiently capture RBD and trimer S from the ancestral strain and the Omicron variant, and the bound targets could be recovered by mild elution at pH 6.0. More importantly, the affinity gel presented excellent and stable chromatographic performance in the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields and purities, and strong HCP reduction. The results demonstrated the potential of SP-HWK as a broad-spectrum peptide ligand for developing a universal platform for the vaccine purification of SARS-CoV-2 and VOCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)