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.

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.

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.

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.

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.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

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.

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.

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena.

BACKGROUND: Antigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA. METHODS: We report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland-Altman (BA) analysis and Lin's concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA. RESULTS: The TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin's CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin's CCC: 0.24) but increased on days 28 and 90 PI (Lins' CCC: 0.88 and 0.98, P < 0.001). CONCLUSIONS/SIGNIFICANCE: The TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.

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.

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.

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.

Planting a chemical flag on antigens.

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

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.

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.

The mechanism of epigallocatechin-3-gallate inhibiting the antigenicity of ß-lactoglobulin under pH 6.2, 7.4 and 8.2: Multi-spectroscopy and molecular simulation methods.

The antigenicity of ß-lactoglobulin (ß-LG) can be influenced by pH values and reduced by epigallocatechin-3-gallate (EGCG). However, a detailed mechanism concerning EGCG decreasing the antigenicity of ß-LG at different pH levels lacks clarity. Here, we explore the inhibition mechanism of EGCG on the antigenicity of ß-LG at pH 6.2, 7.4 and 8.2 using enzyme-linked immunosorbent assay, multi-spectroscopy, mass spectrometry and molecular simulations. The results of Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) elucidate that the noncovalent binding of EGCG with ß-LG induces variations in the secondary structure and conformations of ß-LG. Moreover, EGCG inhibits the antigenicity of ß-LG the most at pH 7.4 (98.30 %), followed by pH 6.2 (73.18 %) and pH 8.2 (36.24 %). The inhibitory difference is attributed to the disparity in the number of epitopes involved in the interacting regions of EGCG and ß-LG. Our findings suggest that manipulating pH conditions may enhance the effectiveness of antigenic inhibitors, with the potential for further application in the food industry.

Subcapsular sinus macrophages maximize germinal center development in non-draining lymph nodes during blood-borne viral infection.

Lymph node (LN) germinal centers (GCs) are critical sites for B cell activation and differentiation. GCs develop after specialized CD169+ macrophages residing in LN sinuses filter antigens (Ags) from the lymph and relay these Ags into proximal B cell follicles. Many viruses, however, first reach LNs through the blood during viremia (virus in the blood), rather than through lymph drainage from infected tissue. How LNs capture viral Ag from the blood to allow GC development is not known. Here, we followed Zika virus (ZIKV) dissemination in mice and subsequent GC formation in both infected tissue-draining and non-draining LNs. From the footpad, ZIKV initially disseminated through two LN chains, infecting LN macrophages and leading to GC formation. Despite rapid ZIKV viremia, non-draining LNs were not infected for several days. Non-draining LN infection correlated with virus-induced vascular leakage and neutralization of permeability reduced LN macrophage attrition. Depletion of non-draining LN macrophages significantly decreased GC B cells in these nodes. Thus, although LNs inefficiently captured viral Ag directly from the blood, GC formation in non-draining LNs proceeded similarly to draining LNs through LN sinus CD169+ macrophages. Together, our findings reveal a conserved pathway allowing LN macrophages to activate antiviral B cells in LNs distal from infected tissue after blood-borne viral infection.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

Anti-viral and anti-tumor vaccines aim to induce cytotoxic CD8+ T cells (CTL) and antibodies. Conserved protein antigens, such as p24 from human immunodeficiency virus, represent promising component for elicitation CTLs, nevertheless with suboptimal immunogenicity, if formulated as recombinant protein. To enhance immunogenicity and CTL response, recombinant proteins may be targeted to dendritic cells (DC) for cross presentation on MHCI, where mannose receptor and/or other lectin receptors could play an important role. Here, we constructed liposomal carrier-based vaccine composed of recombinant p24 antigen bound by metallochelating linkage onto surface of nanoliposomes with surface mannans coupled by aminooxy ligation. Generated mannosylated proteonanoliposomes were analyzed by dynamic light scattering, isothermal titration, and electron microscopy. Using murine DC line MutuDC and murine bone marrow derived DC (BMDC) we evaluated their immunogenicity and immunomodulatory activity. We show that p24 mannosylated proteonanoliposomes activate DC for enhanced MHCI, MHCII and CD40, CD80, and CD86 surface expression both on MutuDC and BMDC. p24 mannosylated liposomes were internalized by MutuDC with p24 intracellular localization within 1 to 3 h. The combination of metallochelating and aminooxy ligation could be used simultaneously to generate nanoliposomal adjuvanted recombinant protein-based vaccines versatile for combination of recombinant antigens relevant for antibody and CTL elicitation.

Do antibody CDR loops change conformation upon binding?

Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.