RESUMEN
Nontoxic substitutes to mycotoxins can facilitate the development of eco-friendly immunoassays. To explore a novel nontoxic substitute to ochratoxin A (OTA), this study screened shark anti-idiotypic variable new antigen receptors (VNARs) against the alpaca anti-OTA nanobody Nb28 through phage display. After four rounds of biopanning of a naïve VNAR phage display library derived from six adult Chiloscyllium plagiosum sharks, one positive clone, namely, P-3, was validated through a phage enzyme-linked immunosorbent assay (phage ELISA). The recombinant anti-idiotypic VNAR AId-V3 was obtained by prokaryotic expression, and the interactions between Nb28 and AId-V3 were investigated via computer-assisted simulation. The affinity of AId-V3 for Nb28 and its heptamer Nb28-C4bpα was measured using Biacore assay. Combining Nb28-C4bpα with AId-V3, a novel direct competitive ELISA (dcELISA) was developed for OTA analysis, with a limit of detection of 0.44 ng/mL and a linear range of 1.77-32.25 ng/mL. The good selectivity, reliability, and precision of dcELISA were confirmed via cross-reaction analysis and recovery experiments. Seven commercial pepper powder samples were tested using dcELISA and validated using high-performance liquid chromatography. Overall, the shark anti-idiotypic VNAR was demonstrated as a promising nontoxic substitute to OTA, and the proposed method was confirmed as a reliable tool for detecting OTA in food.
Asunto(s)
Camélidos del Nuevo Mundo , Ensayo de Inmunoadsorción Enzimática , Ocratoxinas , Tiburones , Anticuerpos de Dominio Único , Ocratoxinas/análisis , Ocratoxinas/inmunología , Tiburones/inmunología , Animales , Camélidos del Nuevo Mundo/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos Antiidiotipos/inmunología , Receptores de Antígenos/inmunologíaRESUMEN
The jawless vertebrates (agnathans/cyclostomes) are ancestral animals comprising lampreys and hagfishes as the only extant representatives. They possess an alternative adaptive immune system (AIS) that uses leucine-rich repeats (LRR)-based variable lymphocyte receptors (VLRs) instead of the immunoglobulin (Ig)-based antigen receptors of jawed vertebrates (gnathostomes). The different VLR types are expressed on agnathan lymphocytes and functionally resemble gnathostome antigen receptors. In particular, VLRB is functionally similar to the B cell receptor and is expressed and secreted by B-like lymphocytes as VLRB antibodies that bind antigens with high affinity and specificity. The potential repertoire scale of VLR-based antigen receptors is believed to be at least comparable to that of Ig-based systems. VLR proteins inherently possess characteristics that render them excellent candidates for biotechnological development, including tractability to recombinant approaches. In recent years, scientists have explored the biotechnological development and utility of VLRB proteins as alternatives to conventional mammalian antibodies. The VLRB antibody platform represents a non-traditional approach to generating a highly diverse repertoire of unique antibodies. In this review, we first describe some aspects of the biology of the AIS of the jawless vertebrates, which recognizes antigens by means of unique receptors. We then summarize reports on the development of VLRB-based antibodies and their applications, particularly those from the inshore hagfish (Eptatretus burgeri) and their potential uses to address microbial diseases in aquaculture. Hagfish VLRB antibodies (we call Ccombodies) are being developed and improved, while obstacles to the advancement of the VLRB platform are being addressed to utilize VLRBs effectively as tools in immunology. VLRB antibodies for novel antigen targets are expected to emerge to provide new opportunities to tackle various scientific questions. We anticipate a greater interest in the agnathan AIS in general and particularly in the hagfish AIS for greater elucidation of the evolution of adaptive immunity and its applications to address microbial pathogens in farmed aquatic animals and beyond.
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Enfermedades de los Peces , Anguila Babosa , Animales , Anguila Babosa/inmunología , Anguila Babosa/genética , Enfermedades de los Peces/inmunología , Inmunidad Adaptativa , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Proteínas de Peces/inmunología , Proteínas de Peces/genéticaRESUMEN
Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. Drawing inspiration from the physiological conditions of sharks and the physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. VNAR phage libraries generated from Japanese topeshark (Hemitriakis japanica) were enriched under these conditions. We then performed NGS analysis and attempted to select clones that were specifically enriched under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.
Asunto(s)
Anticuerpos , Receptores de Antígenos , Tiburones , Animales , Anticuerpos/inmunología , Receptores de Antígenos/inmunología , Tiburones/inmunología , Anticuerpos de Dominio Único/inmunología , JapónRESUMEN
Vγ9Vδ2 T cells can recognize various molecules associated with cellular stress or transformation, providing a unique avenue for the treatment of cancers or infectious diseases. Nonetheless, Vγ9Vδ2 T-cell-based immunotherapies frequently achieve suboptimal efficacies in vivo. Enhancing the cytotoxic effector function of Vγ9Vδ2 T cells is one potential avenue through which the immunotherapeutic potential of this subset may be improved. We compared the use of four pro-inflammatory cytokines on the effector phenotype and functions of in vitro expanded Vγ9Vδ2 T cells, and demonstrated TCR-independent cytotoxicity mediated through CD26, CD16, and NKG2D, which could be further enhanced by IL-23, IL-18, and IL-15 stimulation throughout expansion. This work defines promising culture conditions that could improve Vγ9Vδ2 T-cell-based immunotherapies and furthers our understanding of how this subset might recognize and target transformed or infected cells.
Asunto(s)
Receptores de Antígenos , Linfocitos T , Humanos , Citocinas/metabolismo , Receptores de Antígenos/inmunología , Proliferación Celular , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Cristalografía por Rayos X , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , Tiburones/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Epítopos , Mutación , Filogenia , Unión Proteica , SARS-CoV-2 , Alineación de Secuencia , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Apoptosis/fisiología , Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Proteína 11 Similar a Bcl2/fisiología , Agammaglobulinemia Tirosina Quinasa/deficiencia , Agammaglobulinemia Tirosina Quinasa/fisiología , Animales , Especificidad de Anticuerpos , Autoanticuerpos/sangre , Linfocitos B/enzimología , Linfocitos B/patología , Proteína 11 Similar a Bcl2/deficiencia , División Celular , Células Cultivadas , Hipergammaglobulinemia/inmunología , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos/inmunología , Síndrome de Sjögren/inmunología , Linfocitos T/inmunologíaRESUMEN
Shark is a cartilaginous fish that produces new antigen receptor (IgNAR) antibodies. This antibody is identified with a similar human heavy chain but dissimilar sequences. The variable domain (VNAR) of IgNAR is stable and small in size, these features are desirable for drug discovery. Previous study results revealed the effectiveness of VNAR as a single molecule or a combination molecule to treat diseases both in vivo and in vitro with promising clinical applications. We showed the first evidence of IgNAR alternative splicing from spotted bamboo shark (Chiloscyllium plagiosum), broadening our understanding of the IgNARs characteristics. In this review, we summarize the discoveries on IgNAR with a focus on its advantages for therapeutic development based on its peculiar biochemistry and molecular structure. Proper applications of IgNAR will provide a novel avenue to understand its special presence in cartilaginous fishes as well as designing a number of drugs for undefeated diseases.
Asunto(s)
Anticuerpos , Proteínas de Peces , Receptores de Antígenos , Tiburones/inmunología , Animales , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/farmacología , Proteínas de Peces/química , Proteínas de Peces/inmunología , Proteínas de Peces/farmacología , Receptores de Antígenos/química , Receptores de Antígenos/inmunologíaRESUMEN
Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.
Asunto(s)
Afinidad de Anticuerpos , Antígenos CD/inmunología , Transporte Biológico/inmunología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Receptores de Transferrina/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Bacteriófagos/inmunología , Transporte Biológico/genética , Reacciones Cruzadas , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Receptores de Antígenos/inmunología , Receptores de Antígenos/metabolismo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/farmacocinética , TransfecciónRESUMEN
Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.
Asunto(s)
Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Vertebrados/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Evolución Biológica , Vía Clásica del Complemento , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citocinas/genética , Citocinas/inmunología , Inmunidad Innata , Linfocitos/citología , Linfocitos/inmunología , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Vertebrados/genéticaRESUMEN
Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.
Asunto(s)
Cíclidos/genética , Cíclidos/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Filogenia , Receptores de Antígenos/química , Alineación de Secuencia/veterinaria , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/fisiologíaRESUMEN
Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Asunto(s)
Anticuerpos Monoclonales , Receptores de Antígenos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Antígenos , Epítopos/metabolismo , Dominios Proteicos/inmunología , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , TiburonesRESUMEN
Whitespotted bamboo shark (Chiloscyllium plagiosum) is a demersal cartilaginous fish with an adaptive immune system founded upon immunoglobulins. In this manuscript, we characterize the IgNAR of the whitespotted bamboo shark. A newly discovered alternative splicing form of IgNAR Sec (IgNARshort (ΔC2-C3) Sec) was identified, in which the C1 domain was spliced directly to the C4 domain, the process resulted in a molecule containing three constant domains. However, a single unpaired cysteine remains in the highly flexible hinge region, contributing in the formation of an interchain disulfide bond. Two types of C1 domain were found, and the one lacking a short α-helix showed lower proportion. This finding suggests that short α-helices might be important to the stability of IgNAR. High-throughput sequencing revealed that the percentage of VNAR types significantly vary between the diverse species of sharks. The variable region of IgNAR (the VNAR) with small size and stabilization is a potential candidate for immunotherapeutic agents. The structure and stability analysis in this manuscript may be useful in future biomedical applications.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Tiburones/genética , Tiburones/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Inmunoglobulinas/química , Filogenia , Receptores de Antígenos/químicaRESUMEN
The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage-associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage-specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.
Asunto(s)
Anticuerpos Monoclonales/genética , Citidina Desaminasa/genética , Lampreas/genética , Receptores de Antígenos/genética , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Lampreas/inmunología , Lampreas/metabolismo , Receptores de Antígenos/inmunología , Receptores de Antígenos/metabolismoRESUMEN
Conventional monoclonal antibodies (mAbs) have been widely used in research and diagnostic applications due to their high affinity and specificity. However, multiple limitations, such as large size, complex structure and sensitivity to extreme ambient temperature potentially weaken the performance of mAbs in certain applications. To address this problem, the exploration of new antigen binders is extensively required in relation to improve the quality of current diagnostic platforms. In recent years, a new immunoglobulin-based protein, namely variable domain of new antigen receptor (VNAR) was discovered in sharks. Unlike conventional mAbs, several advantages of VNARs, include small size, better thermostability and peculiar paratope structure have attracted interest of researchers to further explore on it. This article aims to first present an overview of the shark VNARs and outline the characteristics as an outstanding new reagent for diagnostic and therapeutic applications.
Asunto(s)
Anticuerpos Monoclonales , Proteínas de Peces , Receptores de Antígenos , Tiburones/inmunología , Anticuerpos de Cadena Única , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Proteínas de Peces/inmunología , Proteínas de Peces/uso terapéutico , Receptores de Antígenos/inmunología , Receptores de Antígenos/uso terapéutico , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.
Asunto(s)
Proteínas de Artrópodos/inmunología , Vacunas Bacterianas/inmunología , Braquiuros/inmunología , Receptores de Antígenos/inmunología , Vibrio parahaemolyticus/inmunología , Inmunidad Adaptativa , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Braquiuros/microbiología , Clonación Molecular , Femenino , Hemocitos/inmunología , Hemocitos/metabolismo , Inmunización Secundaria , Masculino , Modelos Moleculares , Filogenia , Receptores de Antígenos/química , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Distribución TisularRESUMEN
Endogenous opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (ORs) are expressed on immune cells. Although drugs of misuse appropriate ORs, conflicting reports indicate immunostimulatory and immunosuppressive activity, in that opioid users have elevated infection risk, opioids activate innate immune cells, and opioids attenuate inflammation in murine T cell-mediated autoimmunity models. The i.v. use of drugs transmits bloodborne pathogens, particularly viruses, making the study of CD8+ T cells timely. From a cohort of nonuser controls and methadone users, we demonstrate, via t-Stochastic Neighbor Embedding and k-means cluster analysis of surface marker expression, that chronic opioid use alters human CD8+ T cell subset balance, with notable decreases in T effector memory RA+ cells. Studying global CD8+ T cell populations, there were no differences in expression of OR and several markers of functionality, demonstrating the need for finer analysis. Purified CD8+ T cells from controls respond to opioids ex vivo by increasing cytoplasmic calcium, a novel finding for OR signal transduction, likely because of cell lineage. CD8+ T cells from controls exposed to µ-OR agonists ex vivo decrease expression of activation markers CD69 and CD25, although the same markers are elevated in µ-OR-treated cells from methadone users. In contrast to control cells, T cell subsets from methadone users show decreased expression of CD69 and CD25 in response to TCR stimulus. Overall, these results indicate a direct, selective role for opioids in CD8+ T cell immune regulation via their ability to modulate cell responses through the opioid receptors and TCRs.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Metadona/efectos adversos , Receptores de Antígenos/inmunología , Receptores Opioides/inmunología , Transducción de Señal/inmunología , Trastornos Relacionados con Sustancias/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/patología , Enfermedad Crónica , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lectinas Tipo C/inmunología , Masculino , Trastornos Relacionados con Sustancias/patologíaRESUMEN
A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Sitios de Unión de Anticuerpos , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , Línea Celular , Proliferación Celular , Supervivencia Celular , Humanos , ProteolisisRESUMEN
Can basic science improve the art of vaccinology? Here, we review efforts to understand immune responses with the aim to improve vaccine design and, eventually, to predict the efficacy of human vaccine candidates using the tools of transformed B cells and targeted transgenic mice carrying B cells with antigen receptors specific for microbes of interest.
Asunto(s)
Linfocitos B/inmunología , Receptores de Antígenos/inmunología , Vacunas/química , Vacunas/inmunología , Vacunología/métodos , Animales , HumanosRESUMEN
Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C259, which recognizes the peptide epitope SLLMWITQC presented by HLA-A*02:01. We investigated which amino acids at each position enable a TCR interaction by sequentially replacing every amino acid position outside of anchor positions 2 and 9 with all 19 possible alternative amino acids, resulting in 134 peptides (133 altered peptides plus epitope peptide). Each peptide was individually evaluated using three different in vitro assays: binding of the NY-ESOc259 TCR to the peptide, peptide-dependent activation of TCR-expressing cells, and killing of peptide-presenting target cells. To represent the TCR recognition kernel, we defined Position Weight Matrices (PWMs) for each assay by assigning normalized measurements to each of the 20 amino acids in each position. To predict potential off-target peptides, we applied a novel algorithm projecting the PWM-defined kernel into the human proteome, scoring NY-ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 binding 9-mer peptides. Of the 12 peptides with high predicted score, we confirmed 7 (including NY-ESO-1 antigen SLLMWITQC) strongly activate human primary NY-ESOc259-expressing T cells. These off-target peptides include peptides with up to 7 amino acid changes (of 9 possible), which could not be predicted using the recognition motif as determined by alanine scans. Thus, this replacement scan assay determines the "TCR fingerprint" and, when coupled with the algorithm applied to the database of human 9-mer peptides binding to HLA-A*02:01, enables the identification of potential off-target antigens and the tissues where they are expressed. This platform enables both screening of multiple TCRs to identify the best candidate for clinical development and identification of TCR-specific cross-reactive peptide recognition and constitutes an improved methodology for the identification of potential off-target peptides presented on MHC class I molecules.
Asunto(s)
Bioensayo , Epítopos de Linfocito T/análisis , Activación de Linfocitos , Péptidos/análisis , Receptores de Antígenos/inmunología , Linfocitos T/inmunología , Línea Celular Tumoral , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Células HEK293 , Humanos , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos/genética , Linfocitos T/citologíaRESUMEN
BACKGROUND/AIM: The aim of the current study was to investigate the synergistic efficacy of Robo1 bichimeric antigen receptor-natural killer cell (BiCAR-NK) immunotherapy and 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model. MATERIALS AND METHODS: The orthotopic pancreatic tumor model was established with human pancreatic cancer BxPC-3 cells expressing red fluorescent protein. The mice were treated with 125I seed implantation alone or the combination of 125I seeds with Robo1-specific CAR-NK cells. To assess tumor inhibition, in vivo fluorescence imaging was conducted. 7 Tesla magnetic resonance (7T-MR) scanning was applied to measure the changes in the metabolic profiles of tumor tissues. RESULTS: Tumor size was significantly reduced in the 125I and 125I +CAR-NK treated group compared to the untreated group (p<0.05). The 125I seed +CAR-NK treated group showed significantly higher tumor reduction than 125I seed treatment alone (p<0.05). T1 diffusion weighted imaging (T1DWI) sequence showed that the tumors of the 125I +BiCAR-NK treated group had a significantly higher grey scale value than the tumors from the untreated control and the group treated with 125I seed alone (p<0.05). CONCLUSION: Robo1 specific CAR-NK immunotherapy enhances efficacy of 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model.