Pathogenic Epitopes Research Articles

Bacteriophage M13KE as a nanoparticle platform to display and deliver a pathogenic epitope: Development of an effective porcine epidemic diarrhoea virus vaccine.

Porcine epidemic diarrhoea virus (PEDV) is a porcine enteric coronavirus, outbreaks and epidemics of which have caused huge economic losses to the livestock industry. The disadvantage of existing PEDV vaccines is that the unstable efficacy and high cost limit their widespread use. Therefore, there is an urgent need to develop a recombinant transgenic vaccine candidate for PEDV. In this study, three linear epitopes on the PEDV spike (S) were screened using peptide scanning. The screened epitopes were linked to targeting peptides for lung and intestinal epithelial cells, respectively, and displayed on the M13KE phage to form recombinant phage nanoparticles. Active immunisation experiments showed that a single B-cell epitope delivered by M13KE phage nanoparticles induced the production of specific neutralising antibodies against PEDV in mice. After PEDV stimulation, the immunised mice had significantly higher levels of interferon-γ (IFN-γ) than the control group. Simultaneously, PEDV stimulation caused lymphocyte activation and proliferation in the immunised mice, which is a typical immune response to viral infections. These results suggest that a single linear antigenic epitope delivered by M13KE phage nanoparticles induces significant humoral and cellular immune responses. The constructed recombinant phage nanoparticles are expected to be potential vaccine candidates for PEDV.

DECODE enables high-throughput mapping of antibody epitopes at single amino acid resolution.

Antibodies are extensively used in biomedical research, clinical fields, and disease treatment. However, to enhance the reproducibility and reliability of antibody-based experiments, it is crucial to have a detailed understanding of the antibody's target specificity and epitope. In this study, we developed a high-throughput and precise epitope analysis method, DECODE (Decoding Epitope Composition by Optimized-mRNA-display, Data analysis, and Expression sequencing). This method allowed identifying patterns of epitopes recognized by monoclonal or polyclonal antibodies at single amino acid resolution and predicted cross-reactivity against the entire protein database. By applying the obtained epitope information, it has become possible to develop a new 3D immunostaining method that increases the penetration of antibodies deep into tissues. Furthermore, to demonstrate the applicability of DECODE to more complex blood antibodies, we performed epitope analysis using serum antibodies from mice with experimental autoimmune encephalomyelitis (EAE). As a result, we were able to successfully identify an epitope that matched the sequence of the peptide inducing the disease model without relying on existing antigen information. These results demonstrate that DECODE can provide high-quality epitope information, improve the reproducibility of antibody-dependent experiments, diagnostics and therapeutics, and contribute to discover pathogenic epitopes from antibodies in the blood.

Epitope Hierarchy in Type 1 Diabetes Pathogenesis.

Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells destroying insulin-producing β cells. Identifying the antigenic epitopes targeted by autoreactive T cells is crucial for understanding pathogenesis, detecting biomarkers, and developing immunotherapies. This paper covers T-cell epitopes in T1D, focusing on pre-proinsulin and hybrid insulin peptides (HIPs) as major autoantigens. Substantial evidence highlights epitopes in the insulin B-chain and C-peptide as dominant targets for pathogenic CD4 and CD8 T cells infiltrating the islets. HIPs, formed by proinsulin fragments ligated to other peptides, constitute a novel class of epitopes detected in human and mouse islets. In addition, the paper also examines neoepitopes arising from posttranslational modifications, splice variants, and defective ribosomal products. A key challenge is differentiating genuinely pathogenic epitopes driving disease from nonpathogenic mimotopes. Identifying any essential, indispensable epitopes among this array could enable the development of antigen-specific immunotherapies targeting the root causative factors underlying T1D.

Impact of Epstein-Barr Virus Nuclear Antigen 1 on Neuroinflammation in PARK2 Knockout Mice.

This study aimed to explore the intricate relationship between mitochondrial dysfunction, infection, and neuroinflammation, focusing specifically on the impact of pathogenic epitopes of the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) in a mouse model of mitochondrial dysfunctions. The investigation included female middle-aged PARK2-/- and C57BL/6J wild-type mice immunized with EBNA1386-405 or with active experimental autoimmune encephalomyelitis (EAE) induction by the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. The PARK2-/- mice developed more severe EAE than the wild-type mice. Following immunization with EBNA1386-405, only PARK2-/- exhibited symptoms resembling EAE. During the acute phase, PARK2-/- mice immunized with either MOG35-55 or EBNA1386-405 exhibited a similar infiltration of the T cells and macrophages in the spinal cord and decreased glial fibrillary acidic protein (GFAP) expression in the brain. However, the EBNA1386-405 -immunized PARK2-/- mice showed significantly increased frequencies of CD8a+ T cells and CD11c+ B cells, and distinct cytokine profiles in the periphery compared to the wild-type controls. These findings highlight the role of EBV in exacerbating inflammation, particularly in the context of mitochondrial deficiencies.

Crinophagic granules in pancreatic β cells contribute to mouse autoimmune diabetes by diversifying pathogenic epitope repertoire

Autoimmune attack toward pancreatic β cells causes permanent loss of glucose homeostasis in type 1 diabetes (T1D). Insulin secretory granules store and secrete insulin but are also thought to be tissue messengers for T1D. Here, we show that the crinophagic granules (crinosome), a minor set of vesicles formed by fusing lysosomes with the conventional insulin dense-core granules (DCG), are pathogenic in T1D development in mouse models. Pharmacological inhibition of crinosome formation in β cells delays T1D progression without affecting the dominant DCGs. Mechanistically, crinophagy inhibition diminishes the epitope repertoire in pancreatic islets, including cryptic, modified and disease-relevant epitopes derived from insulin. These unconventional insulin epitopes are largely undetectable in the MHC-II epitope repertoire of the thymus, where only canonical insulin epitopes are presented. CD4+ T cells targeting unconventional insulin epitopes display autoreactive phenotypes, unlike tolerized T cells recognizing epitopes presented in the thymus. Thus, the crinophagic pathway emerges as a tissue-intrinsic mechanism that transforms insulin from a signature thymic self-protein to a critical autoantigen by creating a peripheral-thymic mismatch in the epitope repertoire.

Type XVII Collagen–Specific CD4+ T Cells Induce Bullous Pemphigoid by Producing IL-5

Bullous pemphigoid is an autoimmune subepidermal blistering disease caused by anti-type XVII collagen (COL17) antibodies. Bullous pemphigoid has some immunological features such as eosinophilic infiltration and the deposition of IgE autoantibodies in the skin; however, the mechanism behind such features remains largely unclear. We focused on the autoantigen-specific CD4+ T cells, which are considered to regulate immune response. We established COL17-specific CD4+ T cell lines invitro. Wild-type mice were immunized with synthesized peptides that include a pathogenic epitope of COL17, and lymphocytes were subjected to a limiting dilution assay. We established 5 T cell lines and examined the pathogenicity by transferring them with COL17-primed B cells into Rag-2-/-/COL17-humanized mice that express human COL17 but not mouse COL17 in the skin. Notably, 3 lines induced bullous pemphigoid-like skin changes associated with subepidermal separation and eosinophilic infiltration histologically and the production of anti-COL17 antibodies. The other 2 lines did not induce such phenotypes. RNA-sequencing analysis revealed that T helper 2 cytokines, particularly IL-5, were highly expressed in the pathogenic T-cell lines. Anti-IL-5 antibody administration significantly reduced the skin changes and attenuated the production of autoantibodies. Thus, the production of IL-5 is critical for COL17-specific CD4+ T cells to induce bullous pemphigoid phenotypes invivo.

Ultralong CDR3 cattlebody use biased to switched immunoglobulin isotypes in bovine immune tissues

Abstract Alongside typical immunoglobulin (Ig), cattle express unique ultralong immunoglobulins (UL) that contain a CDR H3 comprised of a β-ribbon stalk and disulfide-linked knob that reaches over 70 amino acids in length. UL Ig exhibit potential "reach" to recessed pathogen epitopes and can provide broadly neutralizing antibodies to elusive antigens. This study explores the relative amounts of UL Ig in pre-switch isotypes (IgM and IgD) versus post-switch isotypes (IgG, IgA and IgE). Rearranged immunoglobulin heavy chain transcripts from primary and secondary lymphoid tissues of three steer were assessed with primers specific for the variable segments used in ultralong and canonical rearrangements as well as specific subisotype constant regions. Relative expression suggests biased selection for UL Ig in germinal centers and use in mucosal tissues of the ruminant gastrointestinal tract. Despite the explosion of studies exploring the immunogenetic diversification and binding possibilities of the UL Ig knob "picobody", our understanding of the use of the role of these antibodies in the Bovid immune system has been much slower. With a more complete understanding of tissue-specific expression patterns and isotype-specific immune responses using these exceptional paratopes, we'll be better poised to exploit them for improved bovine and human health.

Constructing an ELISA for Detection of Anti-Borrelia in Wildlife and Agricultural Animals.

Zoonotic diseases have major impacts on human and animal health, as well as being ecologically significant. Lyme Borreliosis or Lyme disease, caused by infection by pathogenic members of the Borrelia genus, is among these zoonotic diseases. Serology is one of the most accessible means for indirect surveillance of pathogen presence by monitoring the presence, abundance, and type of immune response to the pathogen or pathogen-associated epitopes. Serological surveillance of wild animals is important as wild animals are the primary reservoirs of manyzoonotic diseases. Similarly, serological surveillance of agricultural animals is important due to their economic importance, in addition to animal welfare concerns. However, serology in any non-model animal such as wildlife or agricultural animals is difficult because serology necessarily relies on blood samples from the animals being tested. While companion or laboratory animals are generally sufficiently accustomed to humans that blood samples can be obtained, obtaining blood samples from wild or agricultural animals is more challenging. This initial challenge is compounded by the absence of validated serological tools to evaluate antibody titres in the sera. In this chapter, we provide methods for constructing an ELISA for the detection of anti-Borrelia antibodies in non-model animals, using studies on horses and cows as a proof of principle. The methods focus on the problems specific to non-model animals including obtaining sera, options for determining positive and negative controls without the ability to perform controlled infections, and methods for test optimization and validation.

Simultaneous combined central and peripheral demyelination with acute inflammatory demyelinating polyradiculoneuropathy and short segment myelitis

Combined central and peripheral demyelination (CCPD) has been proposed to describe the occurrence of demyelination in both central and peripheral nervous systems. The aetiopathogenesis of combined demyelination is unknown, but the possibility of a shared pathogenic epitope has been suggested. Although it has been described in paediatric patients, reports of simultaneous onset CCPD with short segment myelitis among adults are scarce. We report a case of simultaneous onset monophasic CCPD in an adult who developed both Guillain-Barre syndrome and short segment spinal cord demyelination that responded to immunomodulation with intravenous immunoglobulins and plasmapheresis. The patient also had evidence of chronic thyroiditis which supports an autoimmune aetiopathogenesis of CCPD. Differentiating this diagnosis from multiple sclerosis has therapeutic implications.

Computational structure-based approach to study chimeric antigens using a new protein scaffold displaying foreign epitopes.

The identification and recombinant production of functional antigens and/or epitopes of pathogens represent a crucial step for the development of an effective protein-based vaccine. Many vaccine targets are outer membrane proteins anchored into the lipidic bilayer through an extended hydrophobic portion making their recombinant production challenging. Moreover, only the extracellular loops, and not the hydrophobic regions, are naturally exposed to the immune system. In this work, the Domain 3 (D3) from Group B Streptococcus (GBS) pilus 2a backbone protein has been identified and engineered to be used as a scaffold for the display of extracellular loops of two Neisseria gonorrhoeae membrane proteins (PorB.1b and OpaB). A computational structure-based approach has been applied to the design of both the scaffold and the model antigens. Once identified the best D3 engineerable site, several different chimeric D3 displaying PorB.1b and OpaB extracellular loops were produced as soluble proteins. Each molecule has been characterized in terms of solubility, stability, and ability to correctly display the foreign epitope. This antigen dissection strategy allowed the identification of most immunogenic extracellular loops of both PorB.1b and OpaB gonococcal antigens. The crystal structure of chimeric D3 displaying PorB.1b immunodominant loop has been obtained confirming that the engineerization did not alter the predicted native structure of this epitope. Taken together, the reported data suggest that D3 is a novel protein scaffold for epitope insertion and display, and a valid alternative to the production of whole membrane protein antigens. Finally, this work describes a generalized computational structure-based approach for the identification, design, and dissection of epitopes in target antigens through chimeric proteins.

DNA Nanostructures: Self‐Adjuvant Carriers for Highly Efficient Subunit Vaccines

AbstractSubunit vaccines based on antigen proteins or epitopes of pathogens or tumors show advantages in immunological precision and high safety, but are often limited by their low immunogenicity. Adjuvants can boost immune responses by stimulating immune cells or promoting antigen uptake by antigen presenting cells (APCs), yet existing clinical adjuvants struggle in simultaneously achieving these dual functions. Additionally, the spatial organization of antigens might be crucial to their immunogenicity. Hence, superior adjuvants should potently stimulate the immune system, precisely arrange antigens, and effectively deliver antigens to APCs. Recently, precisely organizing and delivering antigens with the unique editability of DNA nanostructures has been proposed, presenting unique abilities in significantly improving the immunogenicity of antigens. In this minireview, we will discuss the principles behind using DNA nanostructures as self‐adjuvant carriers and review the latest advancements in this field. The potential and challenges associated with self‐adjuvant DNA nanostructures will also be discussed.

DNA Nanostructures: Self-Adjuvant Carriers for Highly Efficient Subunit Vaccines.

Subunit vaccines based on antigen proteins or epitopes of pathogens or tumors show advantages in immunological precision and high safety, but are often limited by their low immunogenicity. Adjuvants can boost immune responses by stimulating immune cells or promoting antigen uptake by antigen presenting cells (APCs), yet existing clinical adjuvants struggle in simultaneously achieving these dual functions. Additionally, the spatial organization of antigens might be crucial to their immunogenicity. Hence, superior adjuvants should potently stimulate the immune system, precisely arrange antigens, and effectively deliver antigens to APCs. Recently, precisely organizing and delivering antigens with the unique editability of DNA nanostructures has been proposed, presenting unique abilities in significantly improving the immunogenicity of antigens. In this minireview, we will discuss the principles behind using DNA nanostructures as self-adjuvant carriers and review the latest advancements in this field. The potential and challenges associated with self-adjuvant DNA nanostructures will also be discussed.

Pruritic crusted lesions in an 89‐year‐old woman

Pruritic crusted lesions in an 89‐year‐old woman

Monoclonal antibody: future of malaria control and prevention.

Monoclonal antibodies (mAbs) are extremely specialized proteins that are cloned from B cells and bind to pathogen epitopes. There are currently no known prophylactic immune-based strategies or efficient, widespread treatments to stop the spread of malaria. In order to lower the prevalence of malaria and its associated mortality, we need mAbs that are capable of offering immediate passive protection against the disease. mAbs have become more crucial in the treatment or prevention of several other infectious diseases. Recently, mAb development for malaria prevention and control has greatly evolved and widespread use in public health settings is now a possibility.

Pretoria: An effective computational approach for accurate and high-throughput identification of CD8+ t-cell epitopes of eukaryotic pathogens

T-cells recognize antigenic epitopes present on major histocompatibility complex (MHC) molecules, triggering an adaptive immune response in the host. T-cell epitope (TCE) identification is challenging because of the extensive number of undetermined proteins found in eukaryotic pathogens, as well as MHC polymorphisms. In addition, conventional experimental approaches for TCE identification are time-consuming and expensive. Thus, computational approaches that can accurately and rapidly identify CD8+ T-cell epitopes (TCEs) of eukaryotic pathogens based solely on sequence information may facilitate the discovery of novel CD8+ TCEs in a cost-effective manner. Here, Pretoria (Predictor of CD8+ TCEs of eukaryotic pathogens) is proposed as the first stack-based approach for accurate and large-scale identification of CD8+ TCEs of eukaryotic pathogens. In particular, Pretoria enabled the extraction and exploration of crucial information embedded in CD8+ TCEs by employing a comprehensive set of 12 well-known feature descriptors extracted from multiple groups, including physicochemical properties, composition-transition-distribution, pseudo-amino acid composition, and amino acid composition. These feature descriptors were then utilized to construct a pool of 144 different machine learning (ML)-based classifiers based on 12 popular ML algorithms. Finally, the feature selection method was used to effectively determine the important ML classifiers for the construction of our stacked model. The experimental results indicated that Pretoria is an accurate and effective computational approach for CD8+ TCE prediction; it was superior to several conventional ML classifiers and the existing method in terms of the independent test, with an accuracy of 0.866, MCC of 0.732, and AUC of 0.921. Additionally, to maximize user convenience for high-throughput identification of CD8+ TCEs of eukaryotic pathogens, a user-friendly web server of Pretoria (http://pmlabstack.pythonanywhere.com/Pretoria) was developed and made freely available.

Determination of the Predictive Roles and Potentially Pathogenic Antigen Epitopes of α-Enolase Related to the Development of Miscarriage in Females with Autoimmune Thyroiditis.

Autoimmune thyroiditis (AIT) is a common endocrine disease which causes a significantly increased risk of miscarriage. Our recent study has shown that the increased ENO1 autoantibody (ENO1Ab) expression in an experimental AIT mouse model was induced by thyroglobulin (Tg) immunization only. In this study, we explored the potential roles of ENO1Ab in miscarriage occurrence among AIT women, and the specific epitopes of ENO1 targeted by ENO1Ab. A total of 432 euthyroid pregnant participants were selected from the project of Subclinical Hypothyroid during Early Pregnancy, including 48 women with AIT and miscarriage, 96 with miscarriage but no AIT, 96 with AIT but no miscarriage, and 192 without either AIT or miscarriage. The enzyme-linked immunosorbent assay was used to determine the serum levels of total IgG against ENO1 and 18 predicted antigen epitopes of ENO1. The results showed that women with AIT and miscarriage had the highest serum levels of ENO1Ab compared to the other groups. Logistic regression analysis showed that the serum ENO1Ab was an independent risk factor for miscarriage, especially among AIT females. The serum level of total IgG against the predicted epitope peptide 6 (i.e., P6 and aa168-183) of ENO1 was significantly increased in women with AIT and miscarriage when compared with those of both the AIT non-miscarriage group and non-AIT miscarriage group. This pilot study suggests that serum ENO1Ab may have a fair predictive value for AIT-related miscarriage, and the autoantibody specific to P6 epitope may especially be more specifically related to this disorder.

Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen's epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection.

Improved predictions of antigen presentation and TCR recognition with MixMHCpred2.2 and PRIME2.0 reveal potent SARS-CoV-2 CD8+ T-cell epitopes

The recognition of pathogen or cancer-specific epitopes by CD8+ Tcells is crucial for the clearance of infections and the response to cancer immunotherapy. This process requires epitopes to be presented on class I human leukocyte antigen (HLA-I) molecules and recognized by the T-cell receptor (TCR). Machine learning models capturing these two aspects of immune recognition are key to improve epitope predictions. Here, we assembled a high-quality dataset of naturally presented HLA-I ligands and experimentally verified neo-epitopes. We then integrated these data in a refined computational framework to predict antigen presentation (MixMHCpred2.2) and TCR recognition (PRIME2.0). The depth of our training data and the algorithmic developments resulted in improved predictions of HLA-I ligands and neo-epitopes. Prospectively applying our tools to SARS-CoV-2 proteins revealed several epitopes. TCR sequencing identified a monoclonal response in effector/memory CD8+ Tcells against one of these epitopes and cross-reactivity with the homologous peptides from other coronaviruses.

The MHC Motif Atlas: a database of MHC binding specificities and ligands.

The highly polymorphic Major Histocompatibility Complex (MHC) genes are responsible for the binding and cell surface presentation of pathogen or cancer specific T-cell epitopes. This process is fundamental for eliciting T-cell recognition of infected or malignant cells. Epitopes displayed on MHC molecules further provide therapeutic targets for personalized cancer vaccines or adoptive T-cell therapy. To help visualizing, analyzing and comparing the different binding specificities of MHC molecules, we developed the MHC Motif Atlas (http://mhcmotifatlas.org/). This database contains information about thousands of class I and class II MHC molecules, including binding motifs, peptide length distributions, motifs of phosphorylated ligands, multiple specificities or links to X-ray crystallography structures. The database further enables users to download curated datasets of MHC ligands. By combining intuitive visualization of the main binding properties of MHC molecules together with access to more than a million ligands, the MHC Motif Atlas provides a central resource to analyze and interpret the binding specificities of MHC molecules.

Identification of a camelid-derived nanobody as a potential therapeutic agent against Streptococcus agalactiae infection

Streptococcus agalactiae (Group B Streptococcus, GBS) is a severe pathogen that has resulted in enormous economic losses in the global tilapia sector. GBS can be effectively controlled by antibiotics to some extent, but antibiotic resistance limits the application of antibiotics. It is urgent to find eco-friendly novel strategies against GBS. As the smallest antibody, nanobodies (Nbs) are novel effective biologics for treating bacterial and viral infections, as they can identify conserved epitopes of hypervariable pathogens. In the study, GBS-specific Nbs were screened from a naive phage-displayed Nb library, and the anti-GBS activity of Nbs was explored in vivo. The results showed that high-affinity Nbs were obtained from a naive phage-displayed Nb library after three rounds of biopanning. A novel nanobody Nb01 was expressed in the Escherichia coli expression system and purified using the Ni-NTA Agarose column. The results of indirect ELISA showed that purified recombinant protein Nb01 still had a high affinity against GBS. Furthermore, we found that Nb01 could effectively inhibit GBS infection in vivo. The copy numbers of GBS in the zebrafish brains were significantly reduced after treatment with Nb01. The survival rate of GBS-infected zebrafish treated with Nb01 was 70.0%, while it was only 43.3% for the controls. The expression of proinflammatory cytokine genes (tnf-α, il-6 and inf-γ) and Wnt-signaling related genes (wnt2, wnt3a and fzd5) in the GBS-infected zebrafish brain were significantly decreased after treatment with Nb01. In conclusion, Nb01 could protect zebrafish from GBS infection, and these results highlighted the potential therapeutic activity of Nbs against GBS in fish.