Breast milk-mediated exposure to dioxins and antigen in infancy enhances antigen-specific antibody production capacity in adulthood in mice.

The immune system is sensitive to many chemicals. Among dioxin compounds, 2,3,7,8-tetrachlorodizenzo-p-dioxin (TCDD) is the most toxic environmental pollutant. The effects of perinatal maternal exposure to dioxins may persist into childhood. However, there have been no reports to date on the effects of exposure to dioxins during infancy, when the immune organs are developing. Therefore, we investigated the effects of TCDD and antigen exposure during lactation on immune function, especially antibody production capacity, in adult mice. Beginning the day after delivery, lactating mothers were orally administered TCDD or a mixture of TCDD and ovalbumin (OVA) daily for 4 weeks, until the pups were weaned. At 6 weeks of age, progeny mice were orally administered OVA daily for 10 weeks, while non-progeny mice were orally administered OVA or a mixture of TCDD and OVA daily for 10 weeks. Production of serum OVA-specific IgG was examined weekly. The amount of TCDD transferred from the mother to the progeny via breast milk was determined by measuring TCDD in the gastric contents of the progeny. A trend toward increasing IgA titer was observed in TCDD-treated mice, and production of IgE was observed only in progeny whose mothers were treated with TCDD and OVA. The results suggest that exposure to TCDD and OVA in breast milk can affect immune function in newborns.

Predicting TCR sequences for unseen antigen epitopes using structural and sequence features.

T-cell receptor (TCR) recognition of antigens is fundamental to the adaptive immune response. With the expansion of experimental techniques, a substantial database of matched TCR-antigen pairs has emerged, presenting opportunities for computational prediction models. However, accurately forecasting the binding affinities of unseen antigen-TCR pairs remains a major challenge. Here, we present convolutional-self-attention TCR (CATCR), a novel framework tailored to enhance the prediction of epitope and TCR interactions. Our approach utilizes convolutional neural networks to extract peptide features from residue contact matrices, as generated by OpenFold, and a transformer to encode segment-based coded sequences. We introduce CATCR-D, a discriminator that can assess binding by analyzing the structural and sequence features of epitopes and CDR3-ß regions. Additionally, the framework comprises CATCR-G, a generative module designed for CDR3-ß sequences, which applies the pretrained encoder to deduce epitope characteristics and a transformer decoder for predicting matching CDR3-ß sequences. CATCR-D achieved an AUROC of 0.89 on previously unseen epitope-TCR pairs and outperformed four benchmark models by a margin of 17.4%. CATCR-G has demonstrated high precision, recall and F1 scores, surpassing 95% in bidirectional encoder representations from transformers score assessments. Our results indicate that CATCR is an effective tool for predicting unseen epitope-TCR interactions. Incorporating structural insights enhances our understanding of the general rules governing TCR-epitope recognition significantly. The ability to predict TCRs for novel epitopes using structural and sequence information is promising, and broadening the repository of experimental TCR-epitope data could further improve the precision of epitope-TCR binding predictions.

Antigen density and applied force control enrichment of nanobody-expressing yeast cells in microfluidics.

In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics.

Targeted Therapy of Multiple Sclerosis: A Case for Antigen-Specific Tregs.

Multiple sclerosis is an autoinflammatory condition that results in damage to myelinated neurons in affected patients. While disease-modifying treatments have been successful in slowing the progression of relapsing-remitting disease, most patients still progress to secondary progressive disease that is largely unresponsive to disease-modifying treatments. Similarly, there is currently no effective treatment for patients with primary progressive MS. Innate and adaptive immune cells in the CNS play a critical role in initiating an autoimmune attack and in maintaining the chronic inflammation that drives disease progression. In this review, we will focus on recent insights into the role of T cells with regulatory function in suppressing the progression of MS, and, more importantly, in promoting the remyelination and repair of MS lesions in the CNS. We will discuss the exciting potential to genetically reprogram regulatory T cells to achieve immune suppression and enhance repair locally at sites of tissue damage, while retaining a fully competent immune system outside the CNS. In the future, reprogramed regulatory T cells with defined specificity and function may provide life medicines that can persist in patients and achieve lasting disease suppression after one cycle of treatment.

Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy.

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

An integrated technology for quantitative wide mutational scanning of human antibody Fab libraries.

Antibodies are engineerable quantities in medicine. Learning antibody molecular recognition would enable the in silico design of high affinity binders against nearly any proteinaceous surface. Yet, publicly available experiment antibody sequence-binding datasets may not contain the mutagenic, antigenic, or antibody sequence diversity necessary for deep learning approaches to capture molecular recognition. In part, this is because limited experimental platforms exist for assessing quantitative and simultaneous sequence-function relationships for multiple antibodies. Here we present MAGMA-seq, an integrated technology that combines multiple antigens and multiple antibodies and determines quantitative biophysical parameters using deep sequencing. We demonstrate MAGMA-seq on two pooled libraries comprising mutants of nine different human antibodies spanning light chain gene usage, CDR H3 length, and antigenic targets. We demonstrate the comprehensive mapping of potential antibody development pathways, sequence-binding relationships for multiple antibodies simultaneously, and identification of paratope sequence determinants for binding recognition for broadly neutralizing antibodies (bnAbs). MAGMA-seq enables rapid and scalable antibody engineering of multiple lead candidates because it can measure binding for mutants of many given parental antibodies in a single experiment.

Engineering high affinity antigen-binders: Beyond conventional antibodies.

For decades, antibodies have remained the archetypal binding proteins that can be rapidly produced with high affinity and specificity against virtually any target. A conventional antibody is still considered the prototype of a binding molecule. It is therefore not surprising that antibodies are routinely used in basic scientific and biomedical research, analytical workflows, molecular diagnostics etc. and represent the fastest growing sector in the field of biotechnology. However, several limitations associated with conventional antibodies, including stringent requirement of animal immunizations, mammalian cells for expression, issues on stability and aggregation, bulkier size and the overall time and cost of production has propelled evolution of concepts along alternative antigen binders. Rapidly evolving protein engineering approaches and high throughput screening platforms have further complemented the development of myriads of classes of non-conventional protein binders including antibody derived as well as non-antibody based molecular scaffolds. These non-canonical binders are finding use across disciplines of which diagnostics and therapeutics are the most noteworthy.

Prediction of Paratope-Epitope Pairs Using Convolutional Neural Networks.

Antibodies play a central role in the adaptive immune response of vertebrates through the specific recognition of exogenous or endogenous antigens. The rational design of antibodies has a wide range of biotechnological and medical applications, such as in disease diagnosis and treatment. However, there are currently no reliable methods for predicting the antibodies that recognize a specific antigen region (or epitope) and, conversely, epitopes that recognize the binding region of a given antibody (or paratope). To fill this gap, we developed ImaPEp, a machine learning-based tool for predicting the binding probability of paratope-epitope pairs, where the epitope and paratope patches were simplified into interacting two-dimensional patches, which were colored according to the values of selected features, and pixelated. The specific recognition of an epitope image by a paratope image was achieved by using a convolutional neural network-based model, which was trained on a set of two-dimensional paratope-epitope images derived from experimental structures of antibody-antigen complexes. Our method achieves good performances in terms of cross-validation with a balanced accuracy of 0.8. Finally, we showcase examples of application of ImaPep, including extensive screening of large libraries to identify paratope candidates that bind to a selected epitope, and rescoring and refining antibody-antigen docking poses.

Electroporation of mRNA as a Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 106 to approximately 108 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors.

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Probiotic lactic acid bacteria promote anti-tumor immunity through enhanced major histocompatibility complex class I-restricted antigen presentation machinery in dendritic cells.

Lactic acid bacteria (LAB) possess the ability to argument T cell activity through functional modification of antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Nevertheless, the precise mechanism underlying LAB-induced enhancement of antigen presentation in APCs remains incompletely understood. To address this question, we investigated the detailed mechanism underlying the enhancement of major histocompatibility complex (MHC) class I-restricted antigen presentation in DCs using a probiotic strain known as Lactococcus lactis subsp. Cremoris C60. We found that Heat-killed-C60 (HK-C60) facilitated the processing and presentation of ovalbumin (OVA) peptide antigen OVA257-264 (SIINFEKL) via H-2Kb in bone marrow-derived dendritic cells (BMDCs), leading to increased generation of effector CD8+ T cells both in vitro and in vivo. We also revealed that HK-C60 stimulation augmented the activity of 20S immunoproteasome (20SI) in BMDCs, thereby enhancing the MHC class I-restricted antigen presentation machinery. Furthermore, we assessed the impact of HK-C60 on CD8+ T cell activation in an OVA-expressing B16-F10 murine melanoma model. Oral administration of HK-C60 significantly attenuated tumor growth compared to control treatment. Enhanced Ag processing and presentation machineries in DCs from both Peyer's Patches (PPs) and lymph nodes (LNs) resulted in an increased tumor antigen specific CD8+ T cells. These findings shed new light on the role of LAB in MHC class-I restricted antigen presentation and activation of CD8+ T cells through functional modification of DCs.

Lab on a bead with oscillatory centrifugal microfluidics for fast and complete mixing enables fast and accurate biomedical assays.

Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example rapid immunoassay in which the amount of captured target is kinetically determined; determination of the concentration of an enzyme or enzyme substrate; or as the final stage in any procedure that involves a capture reagent when an enzyme reaction is used as the indicator. Rapid mixing and precise timing are however difficult to achieve in point-of-care devices designed for small sample volumes and fast time to result. By using centrifugal microfluidics and transposing the reaction surface from a chamber to a single mm-scale bead we demonstrate an elegant and easily manufacturable solution. Reagents (which may be, for example, an enzyme, enzyme substrate, antibody or antigen) are immobilised on the surface of a single small bead (typically 1-2 mm in diameter) contained in a cylindrical reaction chamber subjected to periodically changing rotational accelerations which promote both mixing and uniform mass-transfer to the bead surface. The gradient of Euler force across the chamber resulting from rotational acceleration of the disc, dΩdisc/dt, drives circulation of fluid in the chamber. Oscillation of Euler force by oscillation of rotational acceleration with period, T, less than that of the hydrodynamic relaxation time of the fluid, folds the fluid streamlines. Movement of the bead in response to the fluid and the changing rotational acceleration provides a dynamically changing chamber shape, further folding and expanding the fluid. Bead rotation and translation driven by fluid flow and disc motion give uniformity of reaction over the surface. Critical parameters for mixing and reaction uniformity are the ratio of chamber radius to bead radius, rchamber/rbead, and the product Trchamber(dΩdisc/dt), of oscillation period and Euler force gradient across the fluid. We illustrate application of the concept using the reaction of horse radish peroxidase (HRP) immobilised on the bead surface with its substrate tetramethylbenzidine (TMB) in solution. Acceleration from rest to break a hydrophobic valve provided precise timing for TMB contact with the bead. Solution uniformity from reaction on the surface of the bead in volumes 20-50 uL was obtained in times of 2.5 s or less. Accurate measurement of the amount of surface-bound HRP by model fitting to the measured kinetics of colour development at 10 s intervals is demonstrated.

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens.

Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist Pam3CSK4 was conjugated to the N-terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting Pam3CSK4-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + Pam3CSK4 groups, Pam3CSK4-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, Pam3CSK4-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.

Comparative transcriptional analysis identifies genes associated with the attenuation of Theileria parva infected cells after long-term in vitro culture.

Autologous administration of attenuated Theileria parva-infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva-infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL, PD-1, TGF-ß and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata-infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva-infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria-infected cells.

Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation.

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Planting a chemical flag on antigens.

Next-generation live vaccines are created by autonomous production of nitrated antigens.