Low-affinity Antibodies Research Articles

Photocrosslinkable, Low-Affinity Affibodies Show Improved Transport and Retention in 3D Tumor Spheroids.

The efficacy of affinity-based treatments for cancer and other diseases is often limited by poor distribution throughout the targeted tissue. Although lower-affinity antibodies will penetrate more uniformly, these often reach lower concentrations because of their rapid clearance from the tissue. To increase retention and improve distribution, we created low-affinity photocrosslinkable affibodies that can diffuse into dense tumor matrices with limited tumor barrier formation and then be photocrosslinked in place to cell receptors to increase retention. In testing with 3D tumor spheroids, the addition of a 50 nM photocrosslinkable affibody showed a similar level of accumulation at the edges of the spheroid but a higher level near the middle of the spheroid than the wild-type (non-photocrosslinkable) affibody. These results show that target affinity affects protein transport in tumor microenvironments and that covalently cross-linking the ligands to cells may improve both their transport and retention.

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the β-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Leptin/Ghrelin Axis and Antibodies Reactive to Both Hormones in Relation to Metabolic Syndrome Components in Mexican Children.

Background: Metabolic syndrome (MetS) in children is a rising health issue that is strongly associated with cardiovascular diseases and type 2 diabetes mellitus development. Low-affinity antibodies reactive to leptin and ghrelin are suggested to regulate hormone stability and function; nevertheless, the role of the leptin/ghrelin axis and antibodies reactive to both hormones in relation to MetS or its components in the pediatric population remains unknown. Methods: Fifty-eight children (7-12 years) were included and categorized according to the presence of one or more criteria for the diagnosis of MetS or according to body mass index. Body composition, biochemical variables, and metabolic risk indexes were determined. Antibodies reactive to leptin and ghrelin were quantified by an in-house enzyme-linked immunosorbent assay test. Ratios of leptin/ghrelin hormones and anti-leptin/anti-ghrelin immune complexes were obtained. Results: The biochemical variables glucose (P = 0.0009), insulin (P = 0.0001), leptin (P = 0.0036), HOMA-IR (homeostatic model assessment for insulin resistance) (P < 0.0001), and plasma atherogenic index (P < 0.0001) were significantly higher in children with two or three components of MetS (MetS 2-3) in comparison to children with none or one component (MetS 0-1). Ratios of leptin/ghrelin (P = 0.0307) and anti-leptin/anti-ghrelin immune complexes (P = 0.0338) were higher in MetS 2-3 group versus MetS 0-1 group. In MetS 2-3 group, both insulin (r = 0.4361, P = 0.0293) and HOMA-IR (r = 0.4761, P = 0.0161) were positively correlated with the leptin/ghrelin hormone ratio. Conclusions: The higher leptin/ghrelin hormone ratio scores observed in MetS 2-3 group, along with their correlation with insulin levels and HOMA-IR, highlight the role of leptin and ghrelin on insulin sensitivity and metabolic regulation. An increased ratio of anti-leptin/anti-ghrelin immune complexes suggests affinity changes in these antibodies that may lead to alterations in hormone function.

Abstract 2530: Advancing ICK assay: Real-time, label-free imaging of lymphocyte subsets

Abstract Background: Immune Cell Killing (ICK) Assay is pivotal in immunology and clinical research, uncovering the intricate connections between the immune system and diseases. Traditional ICK assays utilizing antibody staining for endpoint data generation pose challenges in capturing dynamic interactions. Moreover, antibody labeling for data quantification may compromise sensitivity, especially with low-affinity antibodies or a limited number of cells. The anticipation of label-free lymphocyte subset classification and real-time effector/target cell interaction analysis in one field of view is expected to provide more valuable insights than traditional ICK assays. Previous studies achieved 70-80% accuracy in image-based lymphocyte subset classification using digital holographic microscopy, light scattering, etc. but the cells’ condition during the cell sorting or sample preparation process may affect the lymphocyte subset status, resulting in real morphological discrepancies. Our approach focuses on directly sorting human PBMCs to enable label-free, dynamic interaction analysis of natural immune system responses in one field of view. Method: In this study, PBMC samples from 6 individuals were separated using standard-density gradient centrifugation and labeled with antibodies. Data were acquired using the Holotomographic Microscope HT-X1 (Tomocube Inc.). We employed Densenet 121 as the deep learning model architecture, considering the trade-off between memory consumption and performance. The cross-entropy loss function was utilized, and the AdamW optimizer was chosen for model parameter optimization. Input data consisted of 3D refractive index (RI) data for individual cells, each of sized (20, 54, 54). Individual cell labeling was performed using staining information. Results: An overall accuracy of 93.75% in Human PBMC subtype cell classification was achieved. In stage 1, an accuracy of 97.56% was demonstrated in classifying [CD14, CD15, Others]. Among the cells classified as "Others" in stage 2, [CD3, CD19, CD16&amp;CD56] displayed an accuracy of 93.5%. In stage 3, classifying [CD4, CD8] within the CD3 subtype achieved an accuracy of 90.2%. This classification is presumably based on pattern recognition of the chromosomal landscape in holotomography images. Conclusion: Our research has successfully demonstrated the feasibility of real-time label-free lymphocyte subset classification. This approach enabled the classification of effector cells as live cells in ICK assay and the analysis of effector-target cell dynamic interactions. Further training in the classification of more detailed subtype cells and various cell death types will provide additional insights. The advancement of this technology is expected to play a pivotal role in disease prevention, treatment, vaccine development, medical research, and academic studies. Citation Format: Sanggeun Oh, Jaephil Do, Hyun-Seok Min, Dongmin Ryu, Wei sun Park. Advancing ICK assay: Real-time, label-free imaging of lymphocyte subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2530.

Advancements and prospects of novel biologicals for myasthenia gravis: toward personalized treatment based on autoantibody specificities.

Myasthenia gravis (MG) is an antibody-mediated autoimmune disease with a prevalence of 150-250 cases per million individuals. Autoantibodies include long-lived antibodies against the acetylcholine receptor (AChR), mainly of the IgG1 subclass, and IgG4, produced almost exclusively by short-lived plasmablasts, which are prevalent in muscle-specific tyrosine kinase (MuSK) myasthenia gravis. Numerous investigations have demonstrated that MG patients receiving conventional medication today still do not possess satisfactory symptom control, indicating a substantial disease burden. Subsequently, based on the type of the autoantibody and the pathogenesis, we synthesized the published material to date and reached a conclusion regarding the literature related to personalized targeted therapy for MG. Novel agents for AChR MG have shown their efficacy in clinical research, such as complement inhibitors, FcRn receptor antagonists, and B-cell activating factor (BAFF) inhibitors. Rituximab, a representative drug of anti-CD20 therapy, has demonstrated benefits in treatment of MuSK MG patients. Due to the existence of low-affinity antibodies or unidentified antibodies that are inaccessible by existing methods, the treatment for seronegative MG remains complicated; thus, special testing and therapy considerations are necessary. It may be advantageous to initiate the application of novel biologicals at an early stage of the disease. Currently, therapies can also be combined and individualized according to different types of antibodies. With such a wide range of drugs, how to tailor treatment strategies to patients with various conditions and find the most suitable solution for each MG profile are our necessary and urgent aims.

Abstract A044: Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies

Abstract Background: CD38 is a transmembrane protein that is often overexpressed in multiple myeloma (MM) and other hematologic cancers. The success of anti-CD38 monoclonal antibodies, like daratumumab and isatuximab, in the treatment of MM supports the potential of CD38-targeting therapy candidates in development, including chimeric antigen receptor (CAR) engineered natural killer (NK) cells. Because CD38 is expressed on normal hematopoietic cells and other tissues, it is important to assess the affinity and epitope-specificity of the CD38-targeting moiety to limit potential on-target/off-tumor toxicities. To determine how different biophysical characteristics impact CAR activity, we identified a diverse panel of CD38-specific antibodies, which were shown to display a wide range of affinities and epitopes towards CD38 and converted these clones into CARs to evaluate their expression and target-specific and non-specific activation in a high-throughput (HTP) CD38-knockout (CD38 KO) Jurkat screen. Methods: Antibodies against CD38 were identified through hybridoma screening. Humanized mice were immunized with the extracellular domain of CD38, and lymphocytes from lymph nodes were fused with SP2/0 cells to generate hybridoma cells. Supernatants from single cell sorted hybridoma clones were screened against CD38+ and CD38 KO Jurkat cells and were assessed for affinity and epitope via surface plasmon resonance detection with the Carterra LSA. 96 unique clones were converted into CARs and transduced into CD38 KO Jurkat cells. CAR-expressing Jurkat cells were assayed for CAR expression and activation (via CD69 expression) when cultured overnight in the presence or absence of MM.1R target cells. Seven CARs were selected for additional characterization in primary NK cells. NK cells from healthy donors were expanded on our proprietary NKSTIM cell line, knocked out for CD38 using a CRISPR-Cas system, and transduced with CD38 CAR constructs. CD38 CAR NK-mediated cytotoxicity against MM.1S target cells was assessed by IncuCyte® S3 live cell analysis system. Results: Hybridoma screening identified many unique CD38-specific antibodies, displaying a wide range of affinities and epitopes towards CD38. Despite these biophysical differences, many of these antibodies, when converted to CARs, confer target-specific activity. Multiple epitopes on CD38 may be targeted and even low affinity antibodies were sufficient for target-specific CAR activation. Furthermore, the seven selected CAR candidates that were expressed in CD38 KO donor NK cells displayed potent in vitro target cell killing against MM.1S target cells. Conclusions: In summary, we have identified and characterized a range of CD38-specific antibodies. When converted into CARs, these targeting moieties mediate target-specific activation, despite differences in CD38 epitope and in affinity, demonstrating the utility of HTP CAR screening. Moreover, expression of the top seven CAR candidates in CD38 KO NK cells resulted in potent target cell killing, supporting further pre-clinical evaluation in relevant models. Citation Format: Emily N Kang, Jacinda T Chen, Bing Li, Kate Jamboretz, Nitin Patel, Daniel Bedinger, Kyle T Pratt, Jessica Hsieh, Luxuan TE Buren, Chao Guo, Ivan Chan, James B Trager, Sasha Lazetic. Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A044.

Germinal centers output clonally diverse plasma cell populations expressing high- and low-affinity antibodies

Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization togain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies.Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by &amp;gt;100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Clinical characteristics and treatment outcomes in patients with double-seronegative myasthenia gravis.

Double-seronegative myasthenia gravis (dSNMG) is defined as myasthenia gravis (MG) without detectable or low affinity antibodies to acetylcholine receptor (AChR) and muscle-specific kinase (MuSK). There are limited data on detailed clinical features and outcomes after treatment in dSNMG patients. The aim was to describe the clinical characteristics and outcomes in dSNMG patients based on MG scales. A retrospective study was performed of patients diagnosed with MG who had negative AChR or MuSK antibodies and they were compared with an AChR-positive MG cohort. Correlations were made with data from the first and last clinic visits, between demographics, clinical characteristics, treatment and disease severity, based on the Myasthenia Gravis Foundation of America category, Myasthenia Gravis Impairment Index (MGII), Patient Acceptable Symptom State and simple single question (SSQ). Eighty patients met the inclusion criteria for dSNMG. The baseline MGII and SSQ scores in the dSNMG cohort showed no significant differences from the AChR group (p = 0.94 and p = 0.46). The dSNMG cohort MGII and SSQ scores improved significantly at the last clinical evaluation (p = 0.001 and p = 0.047). The MGII improvement in the AChR cohort was significantly better (p = 0.003). The initial severity of dSNMG based on clinical scores is similar to antibody-positive MG patients. There is significant clinical improvement in dSNMG patients after therapy, measured in the last clinical evaluation. This supports an immune pathophysiology of many dSNMG patients.

Toll-like receptor mediated inflammation directs B cells towards protective antiviral extrafollicular responses

Extrafollicular plasmablast responses (EFRs) are considered to generate antibodies of low affinity that offer little protection from infections. Paradoxically, high avidity antigen-B cell receptor engagement is thought to be the main driver of B cell differentiation, whether in EFRs or slower-developing germinal centers (GCs). Here we show that influenza infection rapidly induces EFRs, generating protective antibodies via Toll-like receptor (TLR)-mediated mechanisms that are both B cell intrinsic and extrinsic. B cell-intrinsic TLR signals support antigen-stimulated B cell survival, clonal expansion, and the differentiation of B cells via induction of IRF4, the master regulator of B cell differentiation, through activation of NF-kB c-Rel. Provision of sustained TLR4 stimulation after immunization shifts the fate of virus-specific B cells towards EFRs instead of GCs, prompting rapid antibody production and improving their protective capacity over antigen/alum administration alone. Thus, inflammatory signals act as B cell fate-determinants for the rapid generation of protective antiviral extrafollicular responses.

Increasing sensitivity of antibody-antigen interactions using photo-cross-linking

Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies that are functionally relevant or highly abundant. Here, we use photo-cross-linking and single-particle electron microscopy to increase antibody detection and unveil epitopes of low-affinity and low-abundance antibodies, leading to a broader structural characterization of polyclonal immune responses. We employed this approach across three different viral glycoproteins and showed increased sensitivity of detection relative to currently used methods. Results were most noticeable in early and late time points of a polyclonal immune response. Additionally, the use of photo-cross-linking revealed intermediate antibody binding states and demonstrated a distinctive way to study antibody binding mechanisms. This technique can be used to structurally characterize the landscape of a polyclonal immune response of patients in vaccination or post-infection studies at early time points, allowing for rapid iterative design of vaccine immunogens.

Aging is associated with accumulated T follicular helper cells with decreased metabolic function

Abstract The aging immune system responds poorly to infection and vaccination, producing reduced numbers of germinal centers, low affinity antibody titers, and reduced B cell memory. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have less expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with decreased CD4+ T conventional cells and accumulated Tfh cells and regulatory T cells, even in naïve mice. Despite increased numbers, our results indicate that aged Tfh have decreased metabolic function, which may have repercussions on germinal center responses. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, we adoptively transferred young, antigen-specific B and CD4+ naïve T cells into aged hosts prior to peptide immunization, which resulted in reduced antigen-specific germinal center B cell and Tfh expansion and reduced antibody titers when compared to young hosts. The results from these experiments emphasize the importance of the lymphoid microenvironment in germinal center initiation and maintenance. We hypothesize that age-associated Tfh accumulation may impair antigen-specific Tfh induction and subsequent germinal center responses. Current studies use live-cell 2-photon microscopy to track the impact of age on lymphocyte motility and Tfh:B interactions, in order to determine the relative contributions of aged APCs, non-specific aged Tfh, and aged regulatory T cells to germinal center deficiency. Research supported by the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377

Dual-Modal Immunosensor Made with the Multifunction Nanobody for Fluorescent/Colorimetric Sensitive Detection of Aflatoxin B1 in Maize

In recent years, dual-modal immunosensors based on synthetic nanomaterials have provided accurate and sensitive detection. However, preparation of nanomaterial probes can be time-consuming, laborious, and not limited to producing inactive and low-affinity antibodies. These challenges can be addressed through the multifunction nanobody without conjugation. In this study, a nanobody-enhanced green fluorescent (Nb26-EGFP) was novel produced with a satisfactory affinity and fluorescent properties. Then, a dual-modal fluorescent/colorimetric immunosensor was constructed using the Nb26-EGFP-gold nanoflowers (AuNFs) composite as a probe, to detect the aflatoxin B1 (AFB1). In the maize matrix, the proposed immunosensor showed high sensitivity with a limit of detection (LOD) of 0.0024 ng/mL and a visual LOD of 1 ng/mL, which is 20-fold and 325-fold compared with the Nb26-EGFP-based single-modal immunosensor and original nanobody Nb26-based immunoassay. The performance of the dual-modal assay was validated by a high-performance liquid chromatography method. The recoveries were between 83.19 and 108.85%, with the coefficients of variation below 9.43%, indicating satisfied accuracy and repeatability. Overall, the novel Nb26-EGFP could be used as the detection probe, and the dual-modal immunosensor could be used as a practical detection method for AFB1 in real samples.

Antibody feedback regulates immune memory after SARS-CoV-2 mRNA vaccination

Feedback inhibition of humoral immunity by antibodies was first documented in 19091. Subsequent studies showed that, depending on the context, antibodies can enhance or inhibit immune responses2,3. However, little is known about how pre-existing antibodies influence the development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity anti-SARS-CoV-2 monoclonal antibodies and subsequently two doses of an mRNA vaccine4–8. We found that the recipients of the monoclonal antibodies produced antigen-binding and neutralizing titres that were only fractionally lower compared than in control individuals. However, the memory B cells of the individuals who received the monoclonal antibodies differed from those of control individuals in that they predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations and showed altered receptor binding domain (RBD) target specificity, consistent with epitope masking. Moreover, only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The mechanism underlying these findings was examined in experiments in mice that showed that germinal centres formed in the presence of the same antibodies were dominated by low-affinity B cells. Our results indicate that pre-existing high-affinity antibodies bias germinal centre and memory B cell selection through two distinct mechanisms: (1) by lowering the activation threshold for B cells, thereby permitting abundant lower-affinity clones to participate in the immune response; and (2) through direct masking of their cognate epitopes. This may in part explain the shifting target profile of memory antibodies elicited by booster vaccinations9.

H3K36 methyltransferase NSD1 is essential for normal B1 and B2 cell development and germinal center formation.

B cells, which consist of two well-defined populations: B1 and B2 cells, which can produce antibodies that are essential for host protection against infections, through virus neutralization, opsonization and antibody-dependent cellular cytotoxicity. Epigenetic modifications, such as DNA methylation and histone modification could regulate immune cell differentiation and functions. In this study, we found a significant reduction of GC response in the B cell specific knockout of H3K36 methyltransferase NSD1 (Mb1-Cre+ NSD1fl/fl, NSD1B KO) mice compared with the wildtype control (Mb1-Cre+ NSD1+/+, NSD1B WT). We also demonstrated reduced production of high-affinity antibody, but increased production of low-affinity antibody in the NSD1B KO mice. Further analysis revealed that loss of NSD1 promoted the development of B1 cells by increasing the expression of Rap1b and Arid3a. In conclusion, our data suggest that NSD1 plays an important role in regulation the development of B1 and B2 cells, and the process of germinal center formation and high-affinity antibody production.

Modulation of porcine alphacoronavirus pathogenesis by antibodies to SARS-CoV-2

BACKGROUND: A variety of antibodies against the SARS-CoV-2 with different functional activities can lead to immune pathology and/or modulation of infection due to antibody-dependent enhancement. An important role in the development of ADE, as exemplified by Dengue fever, is played by low-affinity antibodies, which interacting with the virus, do not neutralize it, but on the contrary forms the virus-antibody complex to immune cells (monocytes/macrophages, B-cells). There are conflicting data on the antibody-dependent enhancement presence in COVID-19 coronavirus infection. Some studies indicate a relationship between the severity of infection in patients infected by SARS-СoV-2 and the level of antibodies to closely related coronaviruses.&#x0D; AIM: The aim of the study was to understand the possibility of interaction between antibodies to SARS-CoV-2 with closely related porcine coronavirus (alphacoronavirus) and their ability to modulate the development of infection in vitro.&#x0D; MATERIALS AND METHODS: In the study serums and leukocytes from patients, who are convalescent after COVID-19 infection in 2020, was used. Antibody titers (IgG/IgM) was checked by commercial kit (VektorBest, Russia), based on ELISA method. In the eukaryotic expression system Chinese hamster ovary cells, monoclonal antibody to SARS-CoV-2 spike protein and its Fab version without Fc domain were obtained. As a model, swine alphacoronavirus- transmissible gastroenteritis virus (TGEV) was used. Replication activity of TGEV was studied in porcine leukocyte cell culture. The ability of this virusto induce the interferon gamma synthesis in leukocytes from convalescents was assessed TigraTest SARS-CoV-2 Enzyme-Linked SPOT analysis (Russia). Virus titration and neutralization assay with sera and monoclonal antibodies were carried out in porcine kidney PK-15 cell line.&#x0D; RESULTS: Serum samples from 43 donors (59% female, 41% male) aged from 21 to 56 was studied for the presence of class M and G antibodies to SARS-CoV-2 by ELISA method. Class M antibodies were found in only one donor. The highest titers of class G antibodies (1:400 to 1:1600) were detected among donors 4050 years old. For four donors with the highest antibody titers, HLA-II type was determined. The DQA1 allele in 3 patients was found to have the *0501 variant, which, according to literature data, is associated with autoimmune thyroid diseases. For the DQB1 allele, two convalescents had exactly the same variants (*0602-8). Among HLA DRB1, all patients had different allele variants (*09, *11, *01,*03,*13,*15,*08,*13).&#x0D; Among the samples, studied by ELISPOT method, in 45,5% of samples a positive T-cell response was noted after stimulation of macrophages with the S peptides of the SARS-CoV-2 protein and in 22.7% of samples in response to the pool of N peptides of the SARS-CoV-2 protein. Interestingly, infection of macrophages from convalescents with the transmissible gastroenteritis virus caused interferon-gamma expression in 31,8% of cases.&#x0D; The monoclonal antibody to the Spike protein SARS-CoV-2 and its Fab variant were not able to neutralize the TGEV. Interestingly, serum samples from 16 donors convalescent after COVID-19 caused virus neutralization in PK-15 cell culture at dilutions of 1:4-1:8.&#x0D; CONCLUSIONS: We have shown that porcine alphacoronavirus induces (in 31.8% of cases) the synthesis of interferon gamma in macrophages of recovered afterCOVID-19 donors, which may indicate cross-recognition of the antigen of a closely related coronavirus. However, monoclonal antibodies against the Spike protein SARS-CoV-2 did not demonstrate to TGEV neutralization. In turn, the neutralization of the TGEV by sera from recovered COVID-19 donors suggests that not only Spike protein, but also other coronavirus antigens can play a significant role in antigenic imprinting and antibody-dependent enhancement of COVID-19.

HIV skews the SARS-CoV-2 B cell response towards an extrafollicular maturation pathway.

HIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. We compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing, and regulatory features. This revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal centre (GC) activity, homing capacity, and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naïve B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2-specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. Despite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. This work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative [grant number 64809]), and the Victor Daitz Foundation.

IL-6, IL-10, sFas, granulysin and indicators of intestinal permeability as early biomarkers for a fatal outcome in COVID-19

The clinical presentation of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranges between mild respiratory symptoms and a severe disease that shares many of the features of sepsis. Sepsis is a deregulated response to infection that causes life-threatening organ failure. During sepsis, the intestinal epithelial cells are affected, causing an increase in intestinal permeability and allowing microbial translocation from the intestine to the circulation, which exacerbates the inflammatory response. Here we studied patients with moderate, severe and critical COVID-19 by measuring a panel of molecules representative of the innate and adaptive immune responses to SARS-CoV-2, which also reflect the presence of systemic inflammation and the state of the intestinal barrier. We found that non-surviving COVID-19 patients had higher levels of low-affinity anti-RBD IgA antibodies than surviving patients, which may be a response to increased microbial translocation. We identified sFas and granulysin, in addition to IL-6 and IL-10, as possible early biomarkers with high sensitivity (>73 %) and specificity (>51 %) to discriminate between surviving and non-surviving COVID-19 patients. Finally, we found that the microbial metabolite d-lactate and the tight junction regulator zonulin were increased in the serum of patients with severe COVID-19 and in COVID-19 patients with secondary infections, suggesting that increased intestinal permeability may be a source of secondary infections in these patients. COVID-19 patients with secondary infections had higher disease severity and mortality than patients without these infections, indicating that intestinal permeability markers could provide complementary information to the serum cytokines for the early identification of COVID-19 patients with a high risk of a fatal outcome.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

SummaryVaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome—or could, alternatively, leverage—the effects of circulating primary antibodies on subsequent naive B cell recruitment.

A High-throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19.

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.