A Diagnostic Challenge: Toxoplasmosis Masquerading as Tonsillar Neoplasm.

This case report elucidates an uncommon manifestation of toxoplasmosis characterized by an ulcerative oropharyngeal lesion and cervical lymphadenopathy, which intriguingly simulated a tonsillar neoplasm. The patient, a 28-year-old immunocompetent woman, reported symptoms such as a persistent sore throat, unilateral neck pain, and otalgia. Despite the initial clinical impressions, a diagnostic left subtotal tonsillectomy revealed no malignancy but marked acute and chronic inflammation. A comprehensive investigation subsequently indicated a recent primary infection with Toxoplasma gondii, as evidenced by the presence of high IgM antibodies and low IgG avidity. This unique case underlines the significance of incorporating toxoplasmosis into the differential diagnosis of oropharyngeal lesions, thereby necessitating a meticulous approach to laboratory testing. Laryngoscope, 134:1741-1743, 2024.

DG-Affinity: predicting antigen-antibody affinity with language models from sequences.

BACKGROUND: Antibody-mediated immune responses play a crucial role in the immune defense of human body. The evolution of bioengineering has led the progress of antibody-derived drugs, showing promising efficacy in cancer and autoimmune disease therapy. A critical step of this development process is obtaining the affinity between antibodies and their binding antigens. RESULTS: In this study, we introduce a novel sequence-based antigen-antibody affinity prediction method, named DG-Affinity. DG-Affinity uses deep neural networks to efficiently and accurately predict the affinity between antibodies and antigens from sequences, without the need for structural information. The sequences of both the antigen and the antibody are first transformed into embedding vectors by two pre-trained language models, then these embeddings are concatenated into an ConvNeXt framework with a regression task. The results demonstrate the superiority of DG-Affinity over the existing structure-based prediction methods and the sequence-based tools, achieving a Pearson's correlation of over 0.65 on an independent test dataset. CONCLUSIONS: Compared to the baseline methods, DG-Affinity achieves the best performance and can advance the development of antibody design. It is freely available as an easy-to-use web server at https://www.digitalgeneai.tech/solution/affinity .

Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice.

Positively selected germinal center B cells (GCBC) can either resume proliferation and somatic hypermutation or differentiate. The mechanisms dictating these alternative cell fates are incompletely understood. We show that the protein arginine methyltransferase 1 (Prmt1) is upregulated in murine GCBC by Myc and mTORC-dependent signaling after positive selection. Deleting Prmt1 in activated B cells compromises antibody affinity maturation by hampering proliferation and GCBC light zone to dark zone cycling. Prmt1 deficiency also results in enhanced memory B cell generation and plasma cell differentiation, albeit the quality of these cells is compromised by the GCBC defects. We further demonstrate that Prmt1 intrinsically limits plasma cell differentiation, a function co-opted by B cell lymphoma (BCL) cells. Consistently, PRMT1 expression in BCL correlates with poor disease outcome, depends on MYC and mTORC1 activity, is required for cell proliferation, and prevents differentiation. Collectively, these data identify PRMT1 as a determinant of normal and cancerous mature B cell proliferation and differentiation balance.

Noncovalent antibody catenation on a target surface greatly increases the antigen-binding avidity.

Immunoglobulin G (IgG) antibodies are widely used for diagnosis and therapy. Given the unique dimeric structure of IgG, we hypothesized that, by genetically fusing a homodimeric protein (catenator) to the C-terminus of IgG, reversible catenation of antibody molecules could be induced on a surface where target antigen molecules are abundant, and that it could be an effective way to greatly enhance the antigen-binding avidity. A thermodynamic simulation showed that quite low homodimerization affinity of a catenator, e.g. dissociation constant of 100 µM, can enhance nanomolar antigen-binding avidity to a picomolar level, and that the fold enhancement sharply depends on the density of the antigen. In a proof-of-concept experiment where antigen molecules are immobilized on a biosensor tip, the C-terminal fusion of a pair of weakly homodimerizing proteins to three different antibodies enhanced the antigen-binding avidity by at least 110 or 304 folds from the intrinsic binding avidity. Compared with the mother antibody, Obinutuzumab(Y101L) which targets CD20, the same antibody with fused catenators exhibited significantly enhanced binding to SU-DHL5 cells. Together, the homodimerization-induced antibody catenation would be a new powerful approach to improve antibody applications, including the detection of scarce biomarkers and targeted anticancer therapies.

Reducing affinity as a strategy to boost immunomodulatory antibody agonism.

Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity-function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.

BTG1 mutation yields supercompetitive B cells primed for malignant transformation.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Significance of antibody numbering systems in the development of antibody engineering.

Immunotherapy has become increasingly popular in recent years for treating a variety of diseases including inflammatory, neurological, oncological, and auto-immune disorders. The significant interest in antibody development is due to the high binding affinity and specificity of an antibody against a specific antigen. Recent advances in antibody engineering have provided a different view on how to engineer antibodies in silico for therapeutic and diagnostic applications. In order to improve the clinical utility of therapeutic antibodies, it is of paramount importance to understand the various molecular properties which impact antigen targeting and its potency. In antibody engineering, antibody numbering (AbN) systems play an important role to identify the complementarity determining regions (CDRs) and the framework regions (FR). Hence, it is crucial to accurately define and understand the CDR, FR and the crucial residues of heavy and light chains that aid in the binding of the antibody to the antigenic site. Detailed understanding of amino acids positions are useful for modifying the binding affinity, specificity, physicochemical features, and half-life of an antibody. In this review, we have summarized the different antibody numbering systems that are widely used in antibody engineering and highlighted their significance. Here, we have systematically explored and mentioned the various tools and servers that harness different AbN systems.

Hallucinating structure-conditioned antibody libraries for target-specific binders.

Antibodies are widely developed and used as therapeutics to treat cancer, infectious disease, and inflammation. During development, initial leads routinely undergo additional engineering to increase their target affinity. Experimental methods for affinity maturation are expensive, laborious, and time-consuming and rarely allow the efficient exploration of the relevant design space. Deep learning (DL) models are transforming the field of protein engineering and design. While several DL-based protein design methods have shown promise, the antibody design problem is distinct, and specialized models for antibody design are desirable. Inspired by hallucination frameworks that leverage accurate structure prediction DL models, we propose the FvHallucinator for designing antibody sequences, especially the CDR loops, conditioned on an antibody structure. Such a strategy generates targeted CDR libraries that retain the conformation of the binder and thereby the mode of binding to the epitope on the antigen. On a benchmark set of 60 antibodies, FvHallucinator generates sequences resembling natural CDRs and recapitulates perplexity of canonical CDR clusters. Furthermore, the FvHallucinator designs amino acid substitutions at the VH-VL interface that are enriched in human antibody repertoires and therapeutic antibodies. We propose a pipeline that screens FvHallucinator designs to obtain a library enriched in binders for an antigen of interest. We apply this pipeline to the CDR H3 of the Trastuzumab-HER2 complex to generate in silico designs predicted to improve upon the binding affinity and interfacial properties of the original antibody. Thus, the FvHallucinator pipeline enables generation of inexpensive, diverse, and targeted antibody libraries enriched in binders for antibody affinity maturation.

Regimen of Coronavirus Disease 2019 Vaccination Influences Extent and Kinetics of Antibody Avidity.

We investigated antibody titers and avidity after heterologous versus homologous coronavirus disease 2019 vaccination over 6 months after the second dose. We found a significantly higher avidity in regimens including at least 1 dose of the adenoviral vector vaccine ChAdOx1-S compared with 2 doses of the mRNA vaccine BNT162b2.

Antibody Avidity and Neutralizing Response against SARS-CoV-2 Omicron Variant after Infection or Vaccination.

Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods: Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17ß by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17ß (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Mechanistic insights into the rational design of masked antibodies.

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.

Model-based inference of neutralizing antibody avidities against influenza virus.

To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n = 45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities (ρ = 0.54, 95% CI = [0.31, 0.70], P < 10-4). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Mechanisms underpinning poor antibody responses to vaccines in ageing.

Vaccines are a highly effective intervention for conferring protection against infections and reducing the associated morbidity and mortality in vaccinated individuals. However, ageing is often associated with a functional decline in the immune system that results in poor antibody production in older individuals after vaccination. A key contributing factor of this age-related decline in vaccine efficacy is the reduced size and function of the germinal centre (GC) response. GCs are specialised microstructures where B cells undergo affinity maturation and diversification of their antibody genes, before differentiating into long-lived antibody-secreting plasma cells and memory B cells. The GC response requires the coordinated interaction of many different cell types, including B cells, T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells and stromal cell subsets like follicular dendritic cells (FDCs). This review discusses how ageing affects different components of the GC reaction that contribute to its limited output and ultimately impaired antibody responses in older individuals after vaccination. An understanding of the mechanisms underpinning the age-related decline in the GC response is crucial in informing strategies to improve vaccine efficacy and extend the healthy lifespan amongst older people.

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

Generation, selection and modification of anti-cardiac troponin I antibodies with high specificity and affinity.

Current diagnosis of acute myocardial infarction involves quantification of circulating cTn levels. This work endeavoured to generate and enhance recombinant antibody fragments targeting various epitopes on the N- and C-terminals of the cTnI molecule, thereby facilitating highly sensitive detection of the troponin molecule. From this approach, two anti-cTnI scFv antibodies were successfully selected using either phage display or structural reformatting of full length anti-cTnI IgG. Their antibody binding affinity was further optimised via chain shuffling and/or site directed mutagenesis, resulting in scFv with heightened sensitivity when compared to the wild-type scFv. If used in conjunction with existing anti-mid fragment cTnI antibodies, these N- and C- terminal-targeting scFvs show high potential for the enhancement of current cTnI detection assays by limiting the effects from cTnI degradation or troponin complex formation.

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem.

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coli Assisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag-Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein-protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.