Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Anti-Drug Antibody Incidence Comparison of Therapeutic Proteins Administered Via Subcutaneous vs. Intravenous Route.

Subcutaneous (SC) administration of therapeutic proteins is perceived to pose higher risk of immunogenicity when compared with intravenous (IV) route of administration (RoA). However, systematic evaluations of clinical data to support this claim are lacking. This meta-analysis was conducted to compare the immunogenicity of the same therapeutic protein by IV and SC RoA. Anti-drug antibody (ADA) data and controlling variables for 7 therapeutic proteins administered by both IV and SC routes across 48 treatment groups were analyzed. RoA was the primary independent variable of interest while therapeutic protein, patient population, adjusted dose, and number of ADA samples were controlling variables. Analysis of variance was used to compare the ADA incidence between IV and SC RoA, while accounting for controlling variables and potential interactions. Subsequently, 10 additional therapeutic proteins with ADA data published for both IV and SC administration were added to the above 7 therapeutic proteins and were evaluated for ADA incidence. RoA had no statistically significant effect on ADA incidence for the initial dataset of 7 therapeutic proteins (p = 0.55). The only variable with a significant effect on ADA incidence was the therapeutic protein. None of the other controlling variables, including their interactions with RoA, was significant. When all data from the 17 therapeutic proteins were pooled, there was no statistically significant effect of RoA on ADA incidence (p = 0.81). In conclusion, there is no significant difference in ADA incidence between the IV and SC RoA, based on analysis of clinical ADA data from 17 therapeutic proteins.

Cytokine/Antibody Fusion Protein Design and Evaluation.

Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.

Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry.

Antibodies are essential research tools whose performance directly impacts research conclusions and reproducibility. Owing to its central role in Alzheimer's disease and other dementias, hundreds of distinct antibody clones have been developed against the microtubule-associated protein Tau and its multiple proteoforms. Despite this breadth of offer, limited understanding of their performance and poor antibody selectivity have hindered research progress. Here, we validate a large panel of Tau antibodies by Western blot (79 reagents) and immunohistochemistry (35 reagents). We address the reagents' ability to detect the target proteoform, selectivity, the impact of protein phosphorylation on antibody binding and performance in human brain samples. While most antibodies detected Tau at high levels, many failed to detect it at lower, endogenous levels. By WB, non-selective binding to other proteins affected over half of the antibodies tested, with several cross-reacting with the related MAP2 protein, whereas the "oligomeric Tau" T22 antibody reacted with monomeric Tau by WB, thus calling into question its specificity to Tau oligomers. Despite the presumption that "total" Tau antibodies are agnostic to post-translational modifications, we found that phosphorylation partially inhibits binding for many such antibodies, including the popular Tau-5 clone. We further combine high-sensitivity reagents, mass-spectrometry proteomics and cDNA sequencing to demonstrate that presumptive Tau "knockout" human cells continue to express residual protein arising through exon skipping, providing evidence of previously unappreciated gene plasticity. Finally, probing of human brain samples with a large panel of antibodies revealed the presence of C-term-truncated versions of all main Tau brain isoforms in both control and tauopathy donors. Ultimately, we identify a validated panel of Tau antibodies that can be employed in Western blotting and/or immunohistochemistry to reliably detect even low levels of Tau expression with high selectivity. This work represents an extensive resource that will enable the re-interpretation of published data, improve reproducibility in Tau research, and overall accelerate scientific progress.

Unleashing the power of antibodies: Engineering for tomorrow's therapy.

Antibodies play a crucial role in host defense against various diseases. Antibody engineering is a multidisciplinary field that seeks to improve the quality of life of humans. In the context of disease, antibodies are highly specialized proteins that form a critical line of defense against pathogens and the disease caused by them. These infections trigger the innate arm of immunity by presenting on antigen-presenting cells such as dendritic cells. This ultimately links to the adaptive arm, where antibody production and maturation occur against that particular antigen. Upon binding with their specific antigens, antibodies trigger various immune responses to eliminate pathogens in a process called complement-dependent cytotoxicity and phagocytosis of invading microorganisms by immune cells or induce antibody-dependent cellular cytotoxicity is done by antibodies. These engineered antibodies are being used for various purposes, such as therapeutics, diagnostics, and biotechnology research. Cutting-edge techniques that include hybridoma technology, transgenic mice, display techniques like phage, yeast and ribosome displays, and next-generation sequencing are ways to engineer antibodies and mass production for the use of humankind. Considering the importance of antibodies in protecting from a diverse array of pathogens, investing in research holds great promise to develop future therapeutic targets to combat various diseases.

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.

Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described. This technique allows investigators to examine histone and co-factor interactions with unintegrated viral DNAs as well as to analyze histone modifications in general or in a kinetic fashion at various time points during viral infection.

Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters.

Background: Characterizing the antibody epitope profiles of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 can aid in elucidating the mechanisms underlying the antibody-mediated immune responses elicited by these vaccines. Methods: This study investigated the distinct antibody epitopes toward the SARS-CoV-2 spike (S) protein targeted after a two-dose primary series of mRNA-1273 followed by a booster dose of mRNA-1273 or a variant-updated vaccine among serum samples from clinical trial adult participants. Results: Multiple S-specific epitopes were targeted after primary vaccination; while signal decreased over time, a booster dose after >6 months largely revived waning antibody signals. Epitope identity also changed after booster vaccination in some subjects, with four new S-specific epitopes detected with stronger signals after boosting than with primary vaccination. Notably, the strength of antibody responses after booster vaccination differed by the exact vaccine formulation, with variant-updated mRNA-1273.211 and mRNA-1273.617.2 booster formulations inducing significantly stronger S-specific signals than a mRNA-1273 booster. Conclusion: Overall, these results identify key S-specific epitopes targeted by antibodies induced by mRNA-1273 primary and variant-updated booster vaccination.

Assessing the feasibility of statistical inference using synthetic antibody-antigen datasets.

Simulation frameworks are useful to stress-test predictive models when data is scarce, or to assert model sensitivity to specific data distributions. Such frameworks often need to recapitulate several layers of data complexity, including emergent properties that arise implicitly from the interaction between simulation components. Antibody-antigen binding is a complex mechanism by which an antibody sequence wraps itself around an antigen with high affinity. In this study, we use a synthetic simulation framework for antibody-antigen folding and binding on a 3D lattice that include full details on the spatial conformation of both molecules. We investigate how emergent properties arise in this framework, in particular the physical proximity of amino acids, their presence on the binding interface, or the binding status of a sequence, and relate that to the individual and pairwise contributions of amino acids in statistical models for binding prediction. We show that weights learnt from a simple logistic regression model align with some but not all features of amino acids involved in the binding, and that predictive sequence binding patterns can be enriched. In particular, main effects correlated with the capacity of a sequence to bind any antigen, while statistical interactions were related to sequence specificity.

State of pneumococcal vaccine immunity.

Like the other invasive encapsulated bacteria, Streptococcus pneumoniae is also covered with a polysaccharide structure. Infants and elderly are most vulnerable to the invasive and noninvasive diseases caused by S. pneumoniae. Although antibodies against polysaccharide capsule are efficient in eliminating S. pneumoniae, the T cell independent nature of the immune response against polysaccharide vaccines renders them weakly antigenic. The introduction of protein conjugated capsular polysaccharide vaccines helped overcome the weak immunogenicity of pneumococcal polysaccharides and decreased the incidence of pneumococcal diseases, especially in pediatric population. Conjugate vaccines elicit T cell dependent response which involve the interaction of specialized CD4+ T cells, called follicular helper T cells (Tfh) with germinal center B cells in secondary lymphoid organs. Despite their improved immunogenicity, conjugate vaccines still need to be administered three to four times in infants during the first 15 month of their life because they mount poor Tfh response. Recent studies revealed fundamental differences in the generation of Tfh cells between neonates and adults. As the portfolio of pneumococcal conjugate vaccines continues to increase, better understanding of the mechanisms of antibody development in different age groups will help in the development of pneumococcal vaccines tailored for different ages.

Expression of Collagen XIII in Tissues of the Thyroid and Orbit With Relevance to Thyroid-Associated Ophthalmopathy.

Purpose: Antibodies against collagen XIII have previously been identified in patients with active thyroid-associated ophthalmopathy (TAO). Although collagen XIII expression has been described in extraocular muscles and orbital fat, its detailed localization in extraocular and thyroid tissues and the connection to autoimmunity for collagen XIII remain unclear. Our objective was to map the potential targets for these antibodies in the tissues of the orbit and thyroid. Methods: We evaluated the expression of collagen XIII in human patient and mouse orbital and thyroid tissues with immunostainings and RT-qPCR using Col13a1-/- mice as negative controls. COL13A1 expression in Graves' disease and goiter thyroid samples was compared with TGF-ß1 and TNF, and these were also studied in human thyroid epithelial cells and fibroblasts. Results: Collagen XIII expression was found in the neuromuscular and myotendinous junctions of extraocular muscles, blood vessels of orbital connective tissue and fat and the thyroid, and in the thyroid epithelium. Thyroid expression was also seen in germinal centers in Graves' disease and in neoplastic epithelium. The expression of COL13A1 in goiter samples correlated with levels of TGF-B1. Upregulation of COL13A1 was reproduced in thyroid epithelial cells treated with TGF-ß1. Conclusions: We mapped the expression of collagen XIII to various locations in the orbit, demonstrated its expression in the pathologies of the Graves' disease thyroid and confirmed the relationship between collagen XIII and TGF-ß1. Altogether, these data add to our understanding of the targets of anti-collagen XIII autoantibodies in TAO.

Antibody-mediated targeting of human microglial leukocyte Ig-like receptor B4 attenuates amyloid pathology in a mouse model.

Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-ß (Aß) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aß and carry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping around Aß plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aß load, mitigated some Aß-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

Vaccine development: obligate intracellular bacteria new tools, old pathogens: the current state of vaccines against obligate intracellular bacteria.

Obligate intracellular bacteria have remained those for which effective vaccines are unavailable, mostly because protection does not solely rely on an antibody response. Effective antibody-based vaccines, however, have been developed against extracellular bacteria pathogens or toxins. Additionally, obligate intracellular bacteria have evolved many mechanisms to subvert the immune response, making vaccine development complex. Much of what we know about protective immunity for these pathogens has been determined using infection-resolved cases and animal models that mimic disease. These studies have laid the groundwork for antigen discovery, which, combined with recent advances in vaccinology, should allow for the development of safe and efficacious vaccines. Successful vaccines against obligate intracellular bacteria should elicit potent T cell memory responses, in addition to humoral responses. Furthermore, they ought to be designed to specifically induce strong cytotoxic CD8+ T cell responses for protective immunity. This review will describe what we know about the potentially protective immune responses to this group of bacteria. Additionally, we will argue that the novel delivery platforms used during the Sars-CoV-2 pandemic should be excellent candidates to produce protective immunity once antigens are discovered. We will then look more specifically into the vaccine development for Rickettsiaceae, Coxiella burnetti, and Anaplasmataceae from infancy until today. We have not included Chlamydia trachomatis in this review because of the many vaccine related reviews that have been written in recent years.

Updated Evaluation of Agalsidase Alfa Enzyme Replacement Therapy for Patients with Fabry Disease: Insights from Real-World Data.

The clinical use of agalsidase alfa as enzyme replacement therapy (ERT) for Fabry disease (FD) has spread since 2001, and a large body of evidence of its effectiveness has been collected. This review presents the clinical and laboratory results achieved with agalsidase alfa, which has been published in the literature. Agalsidase alfa infusion slows down or stops the progression of renal damage, expressed by reduction or stabilization of the annual decline of the glomerular filtration rate; yearly decrease of glomerular filtration rate (slope) sometimes is reduced until its stabilization. ERT prevents or reduces the occurrence of hypertrophic cardiomyopathy or slows the increase over time if it is already present. Moreover, regarding neurological manifestations, ERT improves neuropathic pain and quality of life, and recent data indicated that it may also prevent the burden of cerebrovascular disease. In addition to ERT's clinical benefits, crucial topics like the most appropriate time to start therapy and the role of anti-drug antibodies (ADA) are analyzed. Treatment with agalsidase alfa in patients with FD substantially improves their outcomes and enhances their quality of life in patients with FD.

A live attenuated vaccine to prevent severe neonatal Escherichia coli K1 infections.

Preterm birth is currently the leading cause of neonatal morbidity and mortality. Genetic, immunological and infectious causes are suspected. Preterm infants have a higher risk of severe bacterial neonatal infections, most of which are caused by Escherichia coli an in particular E. coli K1strains. Women with history of preterm delivery have a high risk of recurrence and therefore constitute a target population for the development of vaccine against E. coli neonatal infections. Here, we characterize the immunological, microbiological and protective properties of a live attenuated vaccine candidate in adult female mice and their pups against after a challenge by K1 and non-K1 strains of E. coli. Our results show that the E. coli K1 E11 ∆aroA vaccine induces strong immunity, driven by polyclonal bactericidal antibodies. In our model of meningitis, mothers immunized prior to mating transfer maternal antibodies to pups, which protect newborn mice against various K1 and non-K1 strains of E. coli. Given the very high mortality rate and the neurological sequalae associated with neonatal E. coli K1 meningitis, our results constitute preclinical proof of concept for the development of a live attenuated vaccine against severe E. coli infections in women at risk of preterm delivery.

Utility of accessible SARS-CoV-2 specific immunoassays in vaccinated adults with a history of advanced HIV infection.

Accessible SARS-CoV-2-specific immunoassays may inform clinical management in people with HIV, particularly in case of persisting immunodysfunction. We prospectively studied their application in vaccine recipients with HIV, purposely including participants with a history of advanced HIV infection. Participants received one (n = 250), two (n = 249) or three (n = 42) doses of the BNT162b2 vaccine. Adverse events were documented through questionnaires. Sample collection occurred pre-vaccination and a median of 4 weeks post-second dose and 14 weeks post-third dose. Anti-spike and anti-nucleocapsid antibodies were measured with the Roche Elecsys chemiluminescence immunoassays. Neutralising activity was evaluated using the GenScript cPass surrogate virus neutralisation test, following validation against a Plaque Reduction Neutralization Test. T-cell reactivity was assessed with the Roche SARS-CoV-2 IFNγ release assay. Primary vaccination (2 doses) was well tolerated and elicited measurable anti-spike antibodies in 202/206 (98.0%) participants. Anti-spike titres varied widely, influenced by previous SARS-CoV-2 exposure, ethnicity, intravenous drug use, CD4 counts and HIV viremia as independent predictors. A third vaccine dose significantly boosted anti-spike and neutralising responses, reducing variability. Anti-spike titres > 15 U/mL correlated with neutralising activity in 136/144 paired samples (94.4%). Three participants with detectable anti-S antibodies did not develop cPass neutralising responses post-third dose, yet displayed SARS-CoV-2 specific IFNγ responses. SARS-CoV-2 vaccination is well-tolerated and immunogenic in adults with HIV, with responses improving post-third dose. Anti-spike antibodies serve as a reliable indicator of neutralising activity. Discordances between anti-spike and neutralising responses were accompanied by detectable IFN-γ responses, underlining the complexity of the immune response in this population.

Association of different milk fat content with coronary artery disease and myocardial infarction risk: A Mendelian randomization study.

BACKGROUND: Numerous observational studies have investigated on the correlation of whole, semi-skimmed, and skimmed milk with coronary artery disease (CAD) and myocardial infarction (MI) risk; However, no consensus has been reached and evidence on any causal links between these exposures and outcomes remains unclear. This study aimed to conduct univariate and multivariate Mendelian randomization (MR) analyses, using publicly released genome-wide association study summary statistics (GWAS) from the IEU GWAS database, to ascertain the causal association of milk with various fat content with CAD and MI risk. METHODS: For the exposure data, 29, 15, and 30 single-nucleotide polymorphisms for whole milk, semi-skimmed milk, and skimmed milk, respectively, obtained from 360,806 Europeans, were used as instrumental variables. CAD and MI comprised 141,217 and 395,795 samples, respectively. We used inverse variance weighted (IVW), weighted median, MR-Egger regression, and MR Pleiotropy Residual Sum and Outlier analyses to determine whether pleiotropy and heterogeneity could skew the MR results. Sensitivity tests were conducted to verify the robustness of the results. RESULTS: After adjusting for false discovery rates (FDR), we discovered proof that skimmed milk intake is a genetically predicted risk factor for CAD (odds ratio [OR] = 5.302; 95% confidence interval [CI] 2.261-12.432; P < 0.001; FDR-corrected P < 0.001) and MI (OR = 2.287; 95% CI 1.218-4.300; P = 0.010; FDR-corrected P = 0.009). Most sensitivity assessments yielded valid results. Multivariable MR for CAD and MI produced results consistent with those obtained using the IVW method. There was no causal relationship between whole or semi-skimmed milk, and CAD or MI. CONCLUSION: Our findings indicate that the consumption of skimmed milk may increase the risk of CAD and MI. This evidence may help inform dietary recommendations for preventing cardiovascular disease. Further studies are required to elucidate the underlying mechanisms.

STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells.

Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.